2011_chromatography of membrane proteins and ins

Chromatography of MembraneProteins and Lipoproteins

Lello Zolla∗ and Angelo D’AlessandroUniversity of Tuscia, Viterbo, Italy

1 Introduction 12 Chromatography of Membrane Proteins 2

2.1 Methods for Protein Separation andCharacterization 3

2.2 Separation Depending on theMembrane Protein Category 5

3 Chromatography of Lipoproteins 73.1 Methods of Lipoprotein Separation 7

4 Electrophoresis of Membrane Proteins: ABrief Overview 94.1 Classical Gel-based Approaches 94.2 Native Gel-based Approaches 9

5 Examples of Application 95.1 Experimental Considerations 105.2 Membrane Proteins 115.3 Separation of Lipoproteins 23

6 Current Trends 266.1 Membrane Proteins 266.2 Lipoproteins 29

7 Comparison with Other Methods 29Acknowledgments 32Abbreviations and Acronyms 32Related Articles 33References 34

The available methods for the separation of membraneproteins and lipoproteins are sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS/PAGE), followed byimmunoblotting, isoelectric focusing (IEF), and capillaryelectrophoresis (CE), along with the recently introducedgel-based native techniques (blue native (BN) and clearnative (CN)), and high-performance liquid chromatog-raphy (HPLC). In this article, it is shown that HPLCtechniques, given their wide versatility, relative ease of use,and high resolution, may be considered the most valuable

∗ Update based on original article by Lello Zolla, Encyclopedia ofAnalytical Chemistry, 2000, John Wiley & Sons Ltd.Tel.: +39 0761 357 100; fax: +39 0761 357 630. E-mail address:[email protected]

tool for the characterization of virtually any hydrophobicprotein. Application examples are described, and compar-isons with other methods are discussed. Moreover, HPLCis not a destructive technique, and therefore, proteins, onceseparated, are available for further analytical investiga-tions. Among these techniques, quantitative and qualitativeanalyses of the separated fractions can be obtained throughother biophysical approaches, such as crystallography orstructural spectroscopy. Most of these approaches requirepreliminary protein purification (90% or higher), whichcould be rapidly obtained through preliminary HPLC.

1 INTRODUCTION

The importance of membrane proteins is highlightedby the fact that about one-third of all the genes invarious organisms code for this class of proteins.(1) Overtwo-thirds of all medications exert their effects throughmembrane proteins, which makes them a major targetof pharmacological interest.(2) However, approachestargeting membrane proteins are hampered by unminortechnical challenges. Owing to their lipophilic char-acter, the solubilization and separation of membraneproteins and lipoproteins normally requires the use ofdetergents. Consequently, classical protein purificationstrategies, designed for water-soluble proteins, are oflimited value, and the number of reports dealing withthe separation and characterization of such proteins islimited. Moreover, their hydrophobic nature induces self-association into noncovalent multimers, and therefore,all separative methods require preliminary proceduressuch as tedious sequential gradient ultracentrifugationfor sample preparation, with the risk of affecting theresults. Although the available methods for the finalseparation of these hydrophobic proteins are numerous,their high hydrophobicity and the presence of deter-gents make most of these available methods expensiveand technically demanding.(3) The traditional approachesby SDS/PAGE are not only cumbersome but alsorather ineffective for evaluating differences in smallmolecular masses, and the run times required are verylong, typically more than 20–30 h. More complex gel-based approaches, such as two-dimensional isoelectrofo-cusing/sodium dodecyl sulfate polyacrylamide gel elec-trophoresis (2-D-IEF/SDS-PAGE) hold a great separa-tion potential. Although two-dimensional electophoresis(2-DE) has many benefits, the technique does not lenditself to large-scale, high-throughput proteomic analyses,as not all types of proteins are well resolved in thissystem. Proteins bearing extremes of size, hydropho-bicity, or charge fail to enter the gel and are notor underrepresented.(4) Although membrane proteins

Encyclopedia of Analytical Chemistry, Online 2006–2011 John Wiley & Sons, Ltd.This article is 2011 John Wiley & Sons, Ltd.This article was published in the Encyclopedia of Analytical Chemistry in 2011 by John Wiley & Sons, Ltd.DOI: 10.1002/9780470027318.a1607.pub2

2 PEPTIDES AND PROTEINS

with up to 12 transmembrane α-helices have beenresolved and identified by 2-DE-MS (mass spectrom-etry), most membrane proteins have been resistant to thisapproach.(3)

CE could represent a powerful separation techniquefor lipoproteins, but the number of reports on membraneproteins is still limited. It is not surprising that HPLCtechniques, given their wide versatility, relative ease ofuse, and high resolution, may be considered the mostvaluable tool for the characterization of virtually anyhydrophobic protein.(5) Moreover, HPLC is not a destruc-tive technique, and therefore, proteins, once separated,are available for other analytical investigations, such asby SDS/PAGE, or other biophysical analyses, such ascrystallography or structural spectroscopy. Membraneprotein structural biology is still a largely unconqueredarea, given that approximately 25% of all proteins aremembrane proteins and yet <150 unique structures areavailable. Membrane proteins have proven to be difficultto study owing to their partially hydrophobic surfaces,flexibility, and lack of stability, although recent technicaladvancements make it possible to foresee a rapid expan-sion of the field in the near future.(6) The real deal inthese techniques is the need for preliminary purificationof protein species of interest, which is best and rapidlyobtained with HPLC analyses of complex samples. Inany case, before loading a sample on any HPLC system,a preseparation step for membrane proteins or lipopro-teins, according to their hydrophobic characteristics, mustbe achieved by selective extractions. The pretreatmentof these proteins facilitates subsequent separation andprovides a first guideline for the choice of detergent. Itis a general rule, in fact, that when choosing the deter-gent for the running buffers, one should use the one withwhich the protein was solubilized, if possible.(7) In vitrostudies such as crystallization rely on the successful solu-bilization or reconstitution of membrane proteins, whichgenerally involves the careful selection of solubilizingdetergents and mixed lipid/detergent systems. Methodscurrently available for efficient reconstitution and solubi-lization of membrane proteins involve the use of detergentmicelles, mixed lipid/detergent micelles, and bicelles orliposomes.(8) It is also advisable to use less denaturingdetergents and nonionic detergents for solubilization andlikewise as an additive to the running buffers, in orderto retain the biological activity of proteins. This repre-sents one of the most challenging tasks in membraneprotein research, especially for structural investigationpurposes, in which retaining the stability and function ofthe protein during solubilization, reconstitution, and crys-tallization is fundamental.(8) The presence of detergent inall steps preserves the subsequent tendency toward asso-ciation and aggregation and the possibility of nonspecific

interactions with the support used for chromatographicseparation.(8)

Although lipoproteins and membrane proteins showcommon features, specific protocols for the separation ofeach group of proteins have recently been proposed, andtherefore, they are presented separately.

2 CHROMATOGRAPHY OF MEMBRANEPROTEINS

Methods for the separation and characterization ofmembrane proteins differ depending on the type ofinteraction of the protein with the membrane: proteinsembedded in a lipid bilayer (integral membrane proteins)or proteins associated with membrane structures (periph-eral). In the latter case, usually ionic interactions orhydrogen bonds are involved in the anchorage, andtherefore, the protein may be removed by gentle solu-bilization with buffered salt solution and then analyzed.Dilute sodium hydroxide and sodium hydrogen carbonate(between pH 10 and 12), concentrated salt solutions,and complex-forming substances such as ethylenedi-aminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N ,N ,N ′,N ′-tetraacetic acid (EGTA)are used satisfactorily for their solubilization. In the caseof embedded proteins, they must first be extracted fromthe membrane structures and then isolated. Membraneproteins’ preliminary enrichment is often performed byultracentrifugation in sucrose gradient; lectin affinitychromatography in combination with centrifugation,silica beads, or biotinylation; and interaction with immo-bilized streptavidin.(9) Tandem lectin affinity could alsobe applied as an approach for the enrichment ofmembrane glycoproteins. Repeated freezing and thawingcan be applied to dissolve the structures mechanically,thereby allowing most membrane-associated proteins tobe removed. However, solubilization of the membrane isthe most suitable approach. Solubilization of membraneproteins is carried out through the use of detergents andamphipathic molecules, consisting of a polar head groupand a hydrophobic chain (or tail) and exhibiting uniqueproperties in aqueous solutions in which they sponta-neously form (generally) spherical micellar structures.

Membrane proteins are frequently soluble in micellesformed by amphiphillic detergents. Detergents solubilizemembrane proteins by creating a mimic of the naturallipid bilayer environment normally inhabited by theprotein. Four main categories of detergents are commonlyused, namely, ionic detergents, bile acid salts, nonionicdetergents, and zwitterionic detergents (see the article bySeddon et al.(8) for further details).

Solubilization is also achieved with different detergents,such as combinations of chloroform and methanol, which


CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINS 3

were used to extract hydrophobic chloroplast membraneproteins and could be suitable for membrane proteins ofnon-plant-cells as well. Alternative aqueous two-phasesystems could be used, which employ the detergent DDM(n-dodecyl β-D-maltoside), Triton X-114, or PEG for theselective binding of one or more proteins of interest toone of the incompatible aqueous phases.

Chaotropic reagents such as urea and guani-dine hydrochloride are now used less frequently,while solubilization using detergents is still in prin-ciple more practical. Membrane proteins can alsobe purified into lipid/detergent micelles. Indeed, itis also advisable to use less denaturing deter-gents such as 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) or nonionic detergents suchas n-octyl β-D-glucopyranoside (OD) or n-dodecyl-β-D-maltoside (β-DM) for solubilization.(8) Identification ofmembrane proteins could be further complicated by thelack of tryptic cleavage sites across the transmembranechain fragments. Enzymatic digestion often results inlarge, hydrophobic species, making it difficult to finallyidentify the protein species. Analyses of proteins froma number of proteomic studies of cell membranes havedemonstrated that a significant component of the identi-fied proteins is not predicted to contain transmembraneregions. However, the presence of such proteins maysometimes arise as a result of contamination of themembrane preparations or through real associations.(10)

Through the use of various reagents in different steps,the membrane proteins can be prepared according totheir respective solubility and hydrophobic characteris-tics. The effort necessary for solubilization of the proteinsincreases with growing complexity of the membranestructure. Section 5.2.3 reports a complete solubiliza-tion scheme for the thylakoid membrane of chloroplasts,which contains the photosynthetic apparatus consisting ofa large number of proteins, namely, 40 different proteinsassembled into two main complexes: photosystem I (PSI)and photosystem II (PSII). It is shown that by selec-tive extraction, a preseparation of membrane proteinsaccording to their hydrophobic characteristics is achieved.This in turn allows further separation by use of different,mainly chromatographic and electrophoretic, methods.

2.1 Methods for Protein Separation andCharacterization

In membrane protein investigations, several chromato-graphic methods may be used. Generally speaking, thesemethods are reversed-phase high-performance liquidchromatography (RP-HPLC), affinity chromatography,size-exclusion high-performance liquid chromatography(SE-HPLC), and ion-exchange HPLC, as well as high-performance membrane chromatography.

Two-dimensional high-performance liquid chromatog-raphy (2-D-HPLC) involving orthogonal steps of RP-HPLC and strong cation-exchange high-performanceliquid chromatography (SCX-HPLC) have been intro-duced by John Yates, III, which is known asthe multidimensional protein identification technology(MudPIT).(11) Usually membrane proteins are organizedas multimeric units; therefore, no single technique islikely to be sufficient for the complete characteriza-tion of different proteins. Chromatographic methodsoffer the possibility to combine a range of techniquesin order to obtain the maximum amount of relevantinformation consistent with an efficient use of analyticalresources. It is important, therefore, to select a battery ofcomplementary techniques that will ensure to obtain thenecessary separation. Specific considerations for selectionof an appropriate battery of techniques are described inSection 5.

Conventional gel permeation and ion-exchange chro-matography on soft gel supports, although timeconsuming, are used for the preparation of large quan-tities (10–50 mg) of pure proteins, while the preparationof smaller quantities is usually performed at low pressure(fast protein liquid chromatography (FPLC)). HPLC,usually carried out at high pressure (300–400 psi), hasbeen an essential tool for the quantitative and qualita-tive analyses of proteins. Its success increased when rigidsupports were introduced, which are able to withstandhigh pressures and show chemical stability within theworking range of biological separations. The separationtime decreased dramatically.

The first consideration in defining an HPLC methoddevelopment strategy is to establish if we need proteinswith preserved biological properties or rather we needtheir characterization. Proteins vary widely in theirphysicochemical properties, and these properties canhave a great impact on the conditions necessary forHPLC analysis. Solution stability can differ widelyfrom one protein to another, and for a given protein,the solution stability can be highly dependent on pH,temperature, presence of denaturants or detergents, andother factors. Such factors must be explored as part ofthe separation development process in order to selectappropriate conditions and an appropriate detergent,under which artifactual denaturation is minimized. Otherphysicochemical properties to be considered includethe presence or absence of free thiol groups or boundmetals and the effect of the organic solvent used in RP-HPLC on the secondary or tertiary structure. Finally,the presence of detergent must be taken into accountalong with the possibility of nonspecific interactionswith the support used for chromatographic separation.Furthermore, membrane proteins readily self-associateinto noncovalent multimers. Hence, in some instances



one may wish to determine only covalent relatedsubstances, and it is therefore appropriate to conductthe HPLC separation using conditions that dissociatethe noncovalent multimers, whereas in other instancesmeasurement of the extent of self-association may be anobjective of the separation.

2.1.1 Reversed-phase High-performance LiquidChromatography

RP-HPLC seemingly offers the best resolution over allchromatographic methods for thylakoid membranes. Theadvent of supports designed with reduced hydropho-bicity, such as the C4 column, where proteins interactlightly with the matrix, have allowed for more gentleelution conditions, with a consequently higher recoveryof proteins when applied to the separation of the photo-synthetic apparatus. Clearly, using organic solvents andacids would cause the biological activity of the protein tobe lost. Thus, RP-HPLC is a powerful but gentle methodfor the isolation of membrane proteins, where the mainaim is their identification or the determination of theirprimary structure.

RP-HPLC offers the best resolution of all chromato-graphic methods, although up to now it has been rarelyused for the separation of membrane proteins. This isbecause the membrane proteins can be recovered onlypartly – and sometimes not at all – under the separationconditions usually required in RP-HPLC (elution withacetonitrile or 2-propanol gradient, in the presence of0.1% (v/v) trifluoroacetic acid (TFA)).

However, membrane proteins are not easy to analyzeby electrospray ionization mass spectrometry (ESI-MS)because of the presence of detergents, which have tobe added to the sample in order to improve proteinsolubilization; therefore, detergents have to be removedfor successful ESI-MS analysis since they are known toinhibit ionization during the electrospray process. Oneway to circumvent the problems of solubility and ionsuppression is the on-line hyphenation of RP-HPLC toESI-MS, which not only efficiently removes the detergentfrom the samples but also fractionates the various proteincomponents before their ESI-MS investigation.

Recent studies have demonstrated that monolithiccapillary columns based on polystyrene/divinylbenzene(PS-DVB) allow proteins to be separated with very highseparation efficiency.(12) These columns are particularlysuited to study membrane proteins using a unifiedanalytical platform (see later). Despite the considerablearsenal of available technology, analytical protocolsfor membrane proteins vary greatly, and considerableeffort is expended in customizing these protocols forthe investigation of specific problems, which is why theuse of microbore HPLC and packed capillary HPLC

for characterizing biosynthetic proteins is becomingincreasingly widespread.

2.1.2 High-performance Affinity Chromatography

Analytical affinity provides a powerful means to purifyproteins and other biomolecules, with a basic two-stepretention–chaotropic elution procedure with minimalnonspecific interactions, and their subsequent elution in ahighly purified state and in the native state. Affinity chro-matography for preparative purposes became successfulwhen researchers improved several key features of immo-bilized ligand interactions with eluting macromolecules,namely, accessibility of immobilized ligand, selectivityof ligand interaction with soluble macromolecule, andreversibility of macromolecule binding, which allows theirelution without denaturation. In this regard, importantparameters for a successful affinity sorbent for biosep-arations include mechanical, chemical, and biologicalstability, and also the potential for nonspecific binding.Rigid matrices such as polymeric supports, silica, andcontrolled pore glass (CPG) may suffer from nonspecificbinding and low recovery. Cross-linked beaded agaroseor cellulose offers a good compromise between mechan-ical stability and nonspecific binding. These matrices alsohave good chemical stability within the working range ofbiological separations, and the product recovery is excel-lent. The interested reader is referred to other works fora discussion of core chromatographic matrices.(13)

Once a matrix has been selected, the ligand isimmobilized via a stable covalent bond to avoidprogressive leaching and consequent capacity loss. Themost common procedure to activate agarose is the use ofcyanogen bromide (CNBr), which results in the couplingof an amine group of the ligand or spacer through anisourea bond. Matrices prepared by this procedure cansuffer from the serious drawback of high ligand leaching,while more stable linkages are produced by organicsulfonyl chlorides or epihalohydrins.

Lectin affinity chromatography is largely used forlipoproteins, whereas two steps with protein-specificligands, namely, immunoaffinity (IA) and transition-state ligands, are commonly used for the separationof receptors and enzyme proteins, respectively.(14) Themeaning of the word affinity in the context of proteinseparation has undergone evolutionary changes overthe years. The exploitation of molecular recognitionphenomenon is no longer limited to affinity chromatog-raphy modes. Affinity-based separations today includeprecipitation, membrane-based purification, and two-phase/three-phase extractions. Apart from the affinityligands, which have a biological relationship (in vivo)with the target protein, a variety of other ligands arenow used in the affinity-based separations. These include



dyes, chelated metal ions, and peptides obtained by phagedisplay technology, combinatorial synthesis, ribosomedisplay methods, and systematic evolution of ligands byexponential enrichment.(15) Proteomics also needs affinitychromatography to reduce the complexity of the systembefore performing analysis by electrophoresis and MS.(16)

2.1.3 Size-exclusion High-performance LiquidChromatography

The separation of membrane proteins may be achievedsatisfactorily under nondenaturing conditions by SE-HPLC.(17) In this case, the size of the molecules ormultimeric complexes is the basis of separation, althoughdifferences of 5–10 kDa in molecular weight are notsufficient for a significant separation. Since nonspecificinteractions between the sample and support, andamong different sample components, must be suppressed,sometimes the addition of denaturing agents, such aschaotropic reagents or sodium dodecyl sulfate (SDS), isrequired. Obviously, under these denaturing conditions,the resolution and yield are optimized in SE-HPLC, alongwith good reproducibility of the results, but proteins maybe denatured.

2.1.4 Ion-exchange High-performance LiquidChromatography

Ion-exchange HPLC is based on the different ionicinteractions between proteins and anionic or cationiccharges of the support.(17) Unfortunately, in this typeof chromatography, protein interaction with the supportis usually strong and the conditions used for elutionare often denaturing. The advent of supports based onagarose, which owing to its hydrophilic characteristichas a lower level of nonspecific interactions with thesample, has renewed the use of ion-exchange HPLC forprotein separations. However, in the particular case ofmembrane proteins, although the degree of resolutionof ion-exchange HPLC is surpassed only by RP-HPLC,interaction with the support is still too strong, and othermethods must be chosen, or detergents must be added tothe separation buffers.

2.1.5 Hydrophobic Interaction High-performanceLiquid Chromatography

Hydrophobic interaction HPLC can also be used for theisolation of membrane proteins, even if they are so strongthat the sample can no longer be recovered from thecolumn, even with the use of detergents or, in extremecases, organic solvents.

2.1.6 High-performance Membrane Chromatography

Red cells, biomembrane vesicles, proteoliposomes, andliposomes noncovalently immobilized in gel particlesor beads have been used as stationary phases forbiomembrane affinity analyses and ion-exchange chro-matographic separations. Lipid monolayers coupled tosilica beads have been utilized for membrane proteinpurification in detergent solution. Proteins are adsorbedon the liposome surfaces and subsequently separatedby salt gradient elution on charged liposomes formedand entrapped in gel beads upon detergent depletion bydialysis.(18) Hence, both protein size and charge affect theseparation and the elution time. These techniques, in theparticular case of membrane proteins, have considerablepotential because of their high resolution, short runningtimes, and low nonspecific interactions. For more details,the reader is referred to a review.(19)

2.1.7 Multidimensional Protein IdentificationTechnonology

The archetypal approach, termed MudPIT,(20) pioneeredin the laboratory of John Yates, III, has proven tobe a remarkably effective and robust methodologyfor investigating global changes in protein expressionas a function of development and disease.(21 – 23) Inshotgun proteomics, the peptides derived from thedigest of proteins are separated and analyzed ina liquid phase. This also avoids the difficulty ofextracting hydrophobic proteins from gels. Severalshotgun approaches have been thought to circumventthe low solubility and high hydrophobicity limitationsof membrane proteins by using organic solvents,(24)

organic acids,(24) microwave-assisted acid hydrolysis,(25)

or detergent-containing aqueous solution.(26) MudPITcouples strong cation-exchange (SCX) chromatographyand reversed-phase (RP) chromatography to tandem MSin a single microcapillary column. The chromatographyproceeds in cycles characterized by a progressive increasein salt concentration. This allows blowing peptides ontothe SCX column, which is followed by a gradient ofincreasing hydrophobicity to progressively elute peptidesfrom the RP column into the ion source.

2.2 Separation Depending on the Membrane ProteinCategory

From a didactic point of view, proteins embedded inmembranes may be divided on the basis of their biologicalrole into receptors, glycosylated proteins, channel orcarrier proteins, and structural proteins or those withouta particular function.

The first consideration in defining the developmentstrategy of an HPLC method is to establish whether



the main interest is isolation of the protein that isfunctionally active or its characterization. In the formercase, nondenaturing conditions must be used, whereas inthe latter case, any type of detergent or organic eluentmay be used for optimum separation.

2.2.1 Cellular Receptors

In the case of receptorial proteins, they rarely existin aggregated form and are specific for recognizing aparticular ligand. Thus, nondenaturating size-exclusionchromatography (SEC) may not be used, and affinitychromatography is more indicated because immobilizedligand proteins can be bound specifically and elutedselectively. The natural ligand might be an ideal choicefor binding selectivity but may suffer from a high intrinsicbinding affinity, which could lead to inactivation of thereceptor or ligand because of the harsh conditions neededfor release from the affinity support. Lectin affinitychromatography is largely used for glycoproteins, andIA chromatography is the most widely used method forreceptors.

A more practical ligand may be a monoclonal antibody(MAb) to the receptor, which can be preselected formodest affinity and appropriate binding kinetics (onand off rates). In general, to prepare an IA sorbent,MAb is preferred over polyclonal antibody because theMAb can be obtained once a hybridoma clone hasbeen isolated reproducibly and the appropriate MAbcan be selected with the desired binding propertiesto optimize biomolecular adsorption and elution. Thedesired elution conditions can be incorporated into thescreening procedure to identify the most advantageousMAb. Since the binding constant varies from clone toclone, selection of a clone producing an MAb with adesirable binding constant is necessary. An antibody thatgives a good response in either Western blot or enzyme-linked immunosorbent assay (ELISA) is not necessarilythe best ligand for the IA sorbent. An example ofthis procedure is described in Section 5.2.2. Interactionanalysis using such current tools as optical biosensorscan be used to screen for those MAbs with a goodbalance of sufficiently high affinity and finite off rate (seeSection 6). Many receptors have been separated by IA.(27)

A milestone in this field is the separation of transferrinreceptor from plasma membranes of various mammaliancells.(28) It is known that this protein binds with highaffinity to immobilized diferric transferrin, whereas itsaffinity for apotransferrin is low. Consequently, thecomplex of transferrin and transferrin receptor can bedissociated by chelation of ferric ions after the addition ofchelating reagents. In this way, transferrin receptor canbe eluted from the column under mild conditions.

2.2.2 Carriers or Channel Proteins Translocating Ions

Membrane proteins with the function of carriers orcontaining channels translocating ions across membranesmay be separated and identified by the use of theperfusion planar lipid membrane (BLM) techniquecoupled with an HPLC system. This technique has beendemonstrated to be efficient for the fast identification,isolation, and characterization of transport proteins(porins) in the outer membrane of bacteria andother organisms,(29) but it seems to be of generalvalidity.(30) The method is based on solubilization of themembrane, separation of solubilized membrane proteinsinto fractions (100–150) eluted from an HPLC column,followed by immediate screening on the BLM for channel-forming activity, allowing precise localization of theporin-containing protein peak. The principle of the BLMmethod is simple. A planar lipid membrane interposedbetween two electrodes is predictably dull, showing littlepermeability to ions, no voltage dependence, and nointeresting transport behavior. However, the introductionof a channel-forming protein, previously separated byHPLC, that is spontaneously incorporated into thebilayer dramatically alters the situation, allowing chemicalsubstances to cross the lipid bilayer and giving rise toan ionic current. Furthermore, the opening and closingof this single ionic channel can be easily detected,and modification of the channel properties by voltage,pH, ionic composition, blockers, mutation, and chemicalreagents can also be quantified. Although the BLM isan in vitro system, its validity has been demonstratedrepeatedly through the more recent technique of patchclamping, giving confidence that what is measured usingBLM corresponds qualitatively and often quantitativelyto what is observed in whole cells. BLMs have beenintensively used in the identification and reconstruction ofchannel-forming proteins,(31) to determine the depositionof amyloid proteins observed in Alzheimer’s diseasepathology,(32) and in the study of membrane proteinsof other disease-related bacteria, whose porins orpathogenic toxins might be labile.(30,31)

2.2.3 Glycoproteins

More than half of all human proteins are glycosylated.Glycosylation defines the adhesive properties of glyco-conjugates, and it is largely through glycan–proteininteractions that cell–cell and cell–pathogen contactsoccur. Not surprisingly, considering the central rolethey play in molecular encounters, glycoprotein- andcarbohydrate-based drugs and therapeutics represent amore than $20 billion market. Glycomics, the study ofglycan expression in biological systems, relies on effec-tive analytical techniques for the correlation of glycan



structure with function.(33) Determination of the carbo-hydrate composition, type, and branching pattern is animportant step in understanding the biological functionof glycoproteins and in the development of a recom-binant DNA-derived glycoprotein as a pharmaceutical.Unfortunately, glycoproteins exist in a variety of biolog-ically active forms owing to the nature of the diversityof monosaccharides and the variety of possible linkages.It may be estimated that the linkage possibilities of ahexasaccharide yield a possible 4.76 × 109 structures.

The complexity of carbohydrate structures mandatesthat a variety of analytical methods be used for the studyof these forms.

Lectin affinity chromatography is used to separatemembrane glycoproteins, but a promising new procedureis described in Section 5, which uses HPLC in conjunctionwith ESI-MS as a tool to identify the sites of glycosylationand the general nature of the glycosylation. ESI-MS candetect whether an oligosaccharide is O- or N-linked anddifferentiate between complex, high-mannose, and hybridforms.

MS analysis of glycopeptides is made challenging bythe differing chemical properties of glycans and peptides.For example, although the β-O-GlcNAc modificationoccurs to the Ser/Thr residues of many nuclear andcytoplasmic proteins, it was not detected until fairlyrecently because it is both uncharged and labile.(33) Otherthan by lectin affinity chromatography, glycopeptides mayalso be isolated by hydrophilic interaction solid-phaseextraction or chromatography and graphitized carbonsolid-phase extraction. They may also be enriched basedon their high molecular weight using SEC (for review seethe article by Mirzabekov et al.(31)).

2.2.4 Others

When the experimental goal does not include the analysisof the functional role of the membrane proteins andonly their identification and characterization, RP-HPLCoffers the best resolution of all chromatographic methods,especially when coupled on-line with a mass spectrometer.In Sections 5 and 6, some aspects of this application arepresented and discussed.

3 CHROMATOGRAPHY OFLIPOPROTEINS

The term lipoprotein refers to particles that areheterogeneous with respect to size, hydrated density,and composition. The main function of the lipoproteinsystem is to transport lipids to the surrounding tissues.All are made up of cholesterol, cholesterol esters,

triglycerides, phospholipids, and apoproteins in varyingproportions. Lipoproteins have a globular structurecontaining hydrophobic lipids such as triglycerides andcholesterol esters within the interior core and a peripherycontaining more polar lipids, phospholipids, cholesterol,and proteins.

Owing to their strong heterogeneity, classificationof lipoproteins remains undefined. Hydrated density isthe more common form of lipoprotein classification.Thus, lipoproteins are separated into very low-densitylipoprotein (VLDL), low-density lipoprotein (LDL), andhigh-density lipoprotein (HDL) fractions. In this case,lipoproteins are viewed as particles that are heteroge-neous with respect to their physical properties but homo-geneous with respect to apoliprotein composition. TheHDL, LDL, and VLDL groups show different functions,which results in their having different properties withrespect to atherosclerosis, and therefore it is very impor-tant to have methods available for their separation.(34)

On the other hand, the separation and characterizationof lipoproteins with respect to protein composition isof increasing interest because of the growing evidencethat apolipoproteins are better markers of coronary heartdisease (CHD) than serum cholesterol levels(35) and thatthe apoliprotein distribution is important in diagnosinglipoprotein abnormalities. Table 1 summarizes the phys-ical and chemical properties of the main lipoproteinclasses and their subfractions and also enlists the differenttypes of apoproteins and their isomers.

3.1 Methods of Lipoprotein Separation

As an alternative to ultracentrifuge separation, gelpermeation separation does not require expensive ultra-centrifugation instrumentation and has the advantage ofbeing relatively mild and nondestructive in the separationof lipoprotein particles. Gel permeation chromatography(GPC) and affinity chromatography are used satisfacto-rily for preparative purposes and to separate lipoproteinsaccording to their size and apolipoprotein content,respectively. However, these chromatographic methodshave not gained wide acceptance as routine techniques,requiring long analysis times and excessive dilution ofsamples during separation. In this respect, the develop-ment of rigid supports for gel permeation has led to largeimprovements in speed and resolution. GPC of wholeplasma is now used primarily for analytical rather thanpreparative separation of plasma lipoproteins.

The advent of RP-HPLC resulted in renewed interest inthe chromatographic separation of lipoproteins, offeringthe best performance in terms of speed and resolution ofstructural variants. In particular, the use of rigid supports,which are able to withstand high pressures, decreased theseparation time dramatically to 1 h compared with 20 h by



Table 1 Physical and chemical characteristics of lipoproteins: subfraction classes and apolipoproteins

Lipoprotein class Diameter (nm) Subfractions Major apolipoprotein Apolipoprotein isomers

VLDL 30–70 – apoC, apoB, apoE apoC-I, C-II and C-III(0,1,2), C-IVapoE-1, E-2, E-3, E-4, E-5ApoD

LDL 17–26 5 apoB, apoF apoB100apoB48

HDL 7–9 5 apoA-I, apoA-II, apoE apoA-I1,2,3,4apoA-II and A-IV

the conventional gel permeation method. Furthermore,the separation may be easily automated, allowingunattended analysis of multiple samples. Automation alsoallows on-line monitoring of cholesterol in the separatedLDL and HDL fractions (see later).

Two new emergent chromatographic techniques forlipoprotein separations are hydroxyapatite chromatog-raphy and countercurrent chromatography (CCC).(36)

Their combined use allows a better separation and anal-ysis of HDLs, LDLs, and VLDLs from human serumwithout prior ultracentrifugation (Section 6). However,ultracentrifugation is one of the eligible strategies forlipoprotein separation, often in duplicate steps.(37)

3.1.1 Separation by Particle Size and Subfractions

Gel permeation is normally used to separate lipoproteinson the basis of differences in particle size. It wasinitially performed on cross-linked dextran supports,which yielded poor separations owing to their relativelysmall pore size; conversely, agarose gel, which hasa larger pore size, has been found to yield betterseparations.(38) Usually, the eluting lipoproteins arederivatized postcolumn with an enzymatic cholesterolreagent, and therefore, the mixture is passed througha reaction capillary and detected at 500 nm. Completeseparation is usually achieved in <80 min.(39)

Separation of the main classes of lipoproteins intotheir subfractions is of growing interest since eachsubfraction has a different specific physiological func-tion. By GPC, HDL particles may be divided into fivesubfractions,(40) whereas by heparin affinity chromatog-raphy, HDL may be subfractionated according to theapoE content.(41,42) Five different fractions of oxidativelymodified LDL have been identified through ion-exchangechromatography.(43) The human serum HDL fraction maybe divided into five or six subclasses by using hydroxyap-atite chromatography.(36)

3.1.2 Separation of Apolipoprotein and Isomers

Affinity chromatography allows separating lipoproteinson the basis of their apolipoprotein content through

the use of specific and reversible interactions betweenlipoprotein and bound ligands. Affinity chromatog-raphy of lipoproteins can be performed using group-specific or biospecific ligands. Group-specific ligandsinclude concanavalin A, which binds to apoB-containinglipoproteins,(44) and heparin, which interacts specif-ically with apoB and apoE,(45) while antibodies tospecific apolipoproteins serve as biospecific ligandsin IA chromatography. This technique provides thehighest specificity for the separation and isolationof lipoproteins on the basis of their apolipopro-tein content, while group-specific ligands are moreeconomically convenient, but the separation is lessaccurate.

In addition to affinity chromatography, the high-performance gel permeation, ion-exchange chromatog-raphy, or RP mode may be used to separate apolipopro-teins into their isomers. Unfortunately, in these HPLCsystems, only small amounts of sample (<5 mg) can beapplied to the column. However, the speed (<60 min)and ease of operation make these techniques idealfor the analytical separation and characterization ofapolipoproteins. GPC is often used as a preparativetechnique for isolating apolipoproteins derived fromHDL or VLDL, while separation of isoforms can beachieved by either ion-exchange chromatography or RP-HPLC. Apolipoproteins from HDL and VLDL canbe separated in <60 min on high-performance TSKgel permeation columns.(46) Separations of apolipopro-teins by ion-exchange chromatography are based ondifferences in charge. Therefore, this mode of chro-matography can separate apolipoproteins with differentisoelectric points.(42) In contrast, differences in hydropho-bicity are the basis of separation by RP-HPLC. TheRP columns usually have a lower sample capacity(<100 mg) than those used in gel permeation HPLC.Moreover, the column lifetime is relatively short owing tothe instability of silica-based stationary phases at pHextremes. However, there is growing interest in theuse of RP-HPLC as a rapid and highly efficient tech-nique for monitoring heterogenicity in apolipoproteinstructure.(46)



4 ELECTROPHORESIS OF MEMBRANEPROTEINS: A BRIEF OVERVIEW

New techniques have been recently introduced totackle the major issues affecting classic 2-D-PAGEapproaches. This mainly concerns the study of majorhydrophobic proteins, namely, membrane proteins, andprotein complexes through the use of native gel-based approaches. Proteins rarely function in isolation.Rather, they are organized in functional units that aredifferent in size, the number of interacting partners,and stability, ranging from huge stable ribosomes ornuclear pore domains to small and transient signal trans-duction complexes, such as in the case of the band 3interacting partners in the inner layer of red bloodcell membranes, which involve multiprotein compo-nents regulating metabolic modulation through oxygen-saturation-dependent reversible binding of glycolyticenzymes.(47) Membrane complexes represent physiolog-ical regulatory nuclei in anucleated cells, such as red bloodcells, which are also devoid of other organelles.(48)

Studying these multiprotein complexes and micro-domains provides information about the spatiotemporalorganization of signal transduction or metabolic processeswithin a cell. However, a major part of this informationis lost when cells are lysed and proteins digestedbefore analysis. Because isolated protein complexes havemuch reduced complexity, preliminary isolation of thesecomplexes allows the identification of low copy numberproteins from the complex and connecting them toa particular function.(49) Multiprotein complexes andassociated proteins can be isolated and purified by avariety of techniques such as affinity-based, recombinantpull-downs, liquid chromatography (LC), blue nativepolyacrylamide gel electrophoresis (BN-PAGE), 2-DE/LC/CE, and FFE (free flow electrophoresis) methods,followed by MS analysis.(50 – 52)

4.1 Classical Gel-based Approaches

Classical gel-based approaches, such as IEF, SDS-PAGE,16-BAC-PAGE, and CTAB-PAGE (cetyltrimethylam-monium bromide polyacrylamide gel electrophoresis),are excluded, as they separate proteins under dena-turing conditions.(53) These methods could be exploitedto perform analyses on hydrophobic protein fractions.Indeed, the use of 16-BAC instead of SDS, which is ananionic detergent, is beneficial when performing analyseson membrane proteins, as they are usually character-ized by an alkalic pI. Therefore, solubilization stepswith cationic detergents, such as 16-BAC, dramaticallyimprove separation resolution during electrophoreticseparation. This methodology has been shown to workwell both in combination with acrylamide gels and with

IEF gel strips.(53) As a good alternative for 16-BAC,the compound CTAB can be used, resulting in a similarimprovement in membrane protein recovery.(54)

4.2 Native Gel-based Approaches

One of the eligible gel-based approaches to investigateprotein complexes is BN-PAGE, which has beenintroduced by Schagger and Von Jagow almost 20 yearsago.(55) Before performing BN-PAGE, protein complexesare usually solubilized in digitonin, DDM, or Triton-X100,and Coomassie brilliant blue G-250 is applied to providethe protein complex with a negative charge to enhanceelectrophoretic mobility. Typically, a native acrylamidegradient gel is used in the first dimension to separate thecomplexes based on overall size.

These complexes can subsequently be separatedthrough a second-dimension SDS-PAGE on denaturationof the protein complexes. It has been shown thatmitochondrial supercomplexes with a size of up to 5 MDacan be separated in this fashion.(56,57) This strategyallows separation of protein complexes and identificationof interacting partners as an alternative to immunecoprecipitation.(58) The advantage is that no antibodiesare needed to perform this analysis, although detergent-labile interacting proteins are lost from the complexes.(59)

Application of 2-D-BN/SDS-PAGE to the plateletcytosolic and membrane proteome allowed the identifi-cation of 63 proteins from different complexes, 9 of whichwere identified for the first time in human platelets.(60)

CyDye flurophores could be used to stain the BN-PAGE gels in order to perform quantitative analyses onthe resolved protein complexes in a manner similar to theBN-PAGE analyses on erythrocyte membrane performedby van Gestel et al.(61) Membrane protein complexesof erythrocytes from healthy donors and patients withhemolytic anemia were compared as to individuateprotein biomarkers (in this case, spectrin) to be relatedwith membrane disorders. Unfortunately, CoomassieG-250 interferes with CyDyes. Therefore, clear nativepolyacrylamide gel electrophoresis (CN-PAGE) can bealso performed in the absence of Coomassie G-250 (forproteins with pI < 7), while at the expenses of the capacityto resolve protein complexes as they differentially migratemerely based on their varying intrinsic charge. Mildanionic detergents, such as sodium oxycholate, have beenused to cope with this inconvenience.(62)

5 EXAMPLES OF APPLICATION

As mentioned earlier, the separation of membraneproteins and lipoproteins is presented separately owingto the specificity of the methods set up for the two



main protein categories. However, in both cases, HPLCis one of the most convenient methods to be used, andtherefore, a few considerations specifically relevant to theinstrumentation required are presented.

5.1 Experimental Considerations

In the case of proteins, more than for small organiccompounds, HPLC requires extremely precise controlof the solvent composition because the RP-HPLCelution time is much more dependent on organic solventcomposition. Single-pump, low-pressure mixing HPLCgradient systems are widely used for protein analysis.However, when using solvents that are difficult tomix (e.g. 1-propanol), dual-pump, high-pressure mixingis advantageous because the two solvent streams aredelivered continuously rather than in a segmentedmanner. Moreover, with single-pump operation, thecontinuous sparging of solvents with helium, for degassingpurposes, results in changes to the organic solventcomposition. This problem may be minimized bypresaturation of the helium with organic vapor by passingthe sparge gas through a solvent reservoir containing themobile phase.

With respect to detection, generally either 280- or 214-nm wavelength is employed in protein analysis. Detectionat 214 nm is more sensitive and more generally applicablebecause absorption at this wavelength is due to thepeptide backbone. However, some HPLC mobile-phaseconstituents may interfere at this wavelength (e.g. acetatesalts). Detection at 280 nm is somewhat less sensitivebut can be used to detect most proteins (absorption at280 nm is primarily due to the aromatic amino acid sidechains of tryptophan, phenylalanine, and tyrosine). Insome cases, dual-wavelength or diode-array detectors arerecommended in order to provide more detailed spectralinformation for each peak observed.

In general, the sample solution should be similar incomposition (e.g. pH) to the HPLC mobile phase soas to minimize the effect of the sample injection onthe separation efficiency. One exception to this ruleis RP-HPLC, where the organic solvent compositionof the sample should be less than that of the initialmobile phase in order to focus the injected proteinsat the head of the column. Clearly, the protein musthave adequate stability in the selected solvent, and inmany cases, stability must be enhanced by use of arefrigerated autoinjector. When conducting SE-HPLCfor determining noncovalent multimers, one must avoidthe use of denaturants (e.g. organic solvents) or extremepH conditions that might disturb the equilibrium betweenmonomeric and multimeric forms.(17)

The column is the heart of the HPLC separationsystem. Unfortunately, this is the one component over

which the analyst has the least direct control, excep-tion made for direct control of the temperature of thecolumn. Consequently, it is important to work closelywith the column manufacturer to obtain several indepen-dent column-manufacturing batches and to investigatewhether a particular method performs consistently on anumber of such batches. Whenever possible, an HPLCmethod to be used on a routine basis should be validatedusing two alternative sources (brands) of columns in orderto minimize the impact of column-manufacture problems.

The advent of new RPs such as C-4 allowed excellentseparations of hydrophobic proteins with a high proteinrecovery (see later). Upon approximately 20 years ofstagnation, packed-column technology has begun toevolve rapidly in the past 10 years. The first monolithiccolumns appeared at the turn of the last century,threatening the monopoly of columns packed withparticles but slowly losing momentum. Then, the averageparticle size routinely used in commercial columns beganto decrease progressively, first from 5 to 3 µm and latelyto sub-2-µm particles. Columns packed with particles ofthe latter particle size allowed reaching high-resolutionpower with reduced plate heights as low as 3.2 µm (i.e.Ca 300 000 plates per meter) for small molecules andreduced analysis times by Ca one order of magnitude.The main disadvantage of such columns is the need forsuitable instruments that are capable of operating at apressure as high as 15 000 psi (1000 bar) and to recordwithout significant distortion to the very narrow peakseluted from the columns. In about the same time whensub-2-µm particles were commercialized, other scientistssearched to prepare particles that are large enough to beused at velocities somewhat larger than their optimumvelocity for maximum efficiency and exhibit low masstransfer resistance. This combination can provide columnsthat can be operated with conventional pumps but haveefficiency comparable to that of columns packed withsub-2-µm particles. Nonetheless, C4 and C8 columns canoften be useful where separations are difficult with C18columns.

The use of microbore HPLC and packed capil-lary HPLC for the characterization of biosyntheticproteins has become more widespread. One of the mainadvantages is the lower solvent consumption. Further-more, such techniques are more compatible with high-performance liquid chromatography/mass spectrometry(HPLC/MS) systems, which are now widely availableand offer profound advantages for method develop-ment (Section 6). The majority of the protein-orientedliterature describes efforts for improving resolution ofthe separation and increasing the sequence coverageof a protein by separating complex mixtures of tryp-tically digested proteins. Methods developed, such aspeak parking and multidimensional separations, have



increased the sensitivity of sample analysis and enabledfurther insight into the protein sample. However, sensi-tivity is the driving factor to develop HPLC columns ofsmaller and smaller diameters and to develop new typesof stationary phases, which are capable of selectivelybinding certain types of analyte. Theoretically, reducingthe internal diameter (ID) of a column from d1 to d2 resultsin an enhanced sensitivity (f ), given by f = d1

2/d22. This

means that a change in column ID from 300 to 75 µmwould result in a 16-fold increase in sensitivity or, moreobviously, the change from a conventional 4.6-mm- to 75-µm-ID column would result in a (theoretical) sensitivitygain of more than 3600-fold.(63)

Taken together, these reasons contributed to makethe nano-HPLC-MS workflow the strategy of choicefor identification of proteins from biological mixtures,especially upon preliminary separation steps involvinggel-based approaches (either 1-D-SDS-PAGE or 2-D-GE(gel electrophoresis)).

5.2 Membrane Proteins

Here three examples are presented.

5.2.1 Cellular Receptor

CD4 is an integral membrane glycoprotein of Tcells, which acts as the cellular receptor for humanimmunodeficiency virus (HIV). Many protocols havebeen set up to recognize and separate CD4. All arebased on IA using MAbs and autoantibodies(64) directedagainst conformational epitopes of CD4.

5.2.1.1 Selecting a Monoclonal Antibody SolubleCD4 (sCD4) had been considered as a possible thera-peutic agent for acquired immune deficiency syndrome(AIDS) by acting as a molecular decoy, i.e. by bindingto the gp120 coat protein of HIV and thereby preventingcellular binding of HIV. Consequently, there has been agrowing interest in searching CD4 variants from eithermammalian cell culture or microbial extracts, showingimproved pharmacokinetic properties. A rapid genericpurification scheme for sCD4 constructs has been devel-oped using IA separation. To prepare a robust IA sorbentfor the purification of sCD4, a number of sCD4 mutantsand a series of MAbs were examined. Five differentcommercial anti-CD4 MAbs were immobilized with theprotein amino groups on to a Sepharose matrix containingan 11-atom spacer using active ester chemistry, and allthe sorbents were evaluated individually on a small testcolumn. Figure 1 shows representative chromatograms toillustrate that different antibodies bind CD4 differently.

In the cases of L-92.5 and L-83 clones, the bindingwas restrictively tight, whereas L-71, L-77, and L-104.5

Abs

orba

nce

(280

nm

)

Elution volume

Unboundpeak

Elutedpeak

L-71

L-83

L-92.5

LigandLoad (µg)UnboundEluted

L-7114001062336

L-8314001306

55

L-92.5140082061

Figure 1 Absorbance (A280) profiles of sCD4 binding to IAcolumn. sCD4, at 0.1 mg mL−1 in N-(2-hydroxyethyl)pipera-zine-N-(2-ethanesulfonic acid) (HEPES) buffer at pH 7.5, wasloaded at 60 cm h−1 at 4 °C on to a 1- × 6.4-cm IA column,preequilibrated with 0.05 M HEPES, 0.15 M NaCl, 0.01% PEG3400 (pH 7.5). To remove nonspecifically bound proteins, thecolumn buffer was washed with two column volumes of HEPEScontaining 0.5 M NaCl at the same flow rate. Bound sCD4 waseluted with 0.1 M acetic acid, 0.15 M NaCl, 0.01% PEG 3400at 60 cm h−1. The inset shows the quantities of sCD4 in variousfractions determined by Bio-Rad protein determination.(65)

(Reproduced by permission of Elsevier Science from C. Jones,A. Patel, S. Griffin, J. Martin, P. Young, K. O’Donnell, C.Silverman, T. Porter, I. Chaiken, J. Chromatogr. A, 707, 3–22(1995).)

showed moderate and more tractable binding affinity.Although L-92.5 sorbent bound the highest amount ofCD4, the recovery was the least of all the sorbents.On the other hand, the L-71 sorbent showed moderatebinding, but the recovery of sCD4 was quantitative. Thus,MAb L-71 was judged to be the most suitable candidatefor immobilization to prepare an affinity sorbent to purifyCD4 and a scale-up IA sorbent to purify CD4 congeners.Using this antibody as IA sorbent, recombinant proteinsloaded on to the column were recovered with very highyield using elution with 0.1 M acetic acid. The proteinsrecovered formed an electrophoretically homogeneousproduct and after neutralization were highly active withrespect to gp120 binding, as judged by a radioligand-beadbinding assay. The sorbent was also used successfully topurify full-length CD4 in highly active form.

A large-scale IA purification process was developed forthe isolation of recombinant sCD4 from Escherichia colifermentations.(66) The MAb used for IA purification ofsCD4 recognized a conformation-dependent epitope onthe surface of the domain 1 of CD4. IA chromatographywas used to purify both sCD4–183, consisting of 183 N-terminal amino acids of human CD4, and sCD4–PE40, a



fusion protein consisting of 178 N-terminal amino acidsof CD4 and amino acids 1–3 and 253–613 of Pseu-domonas exotoxin A (PE40). Relatively high recoveriesof sCD4–183 and sCD4–PE40 were observed in the IAstep of the purification process (71% and 79% recovery,respectively). sCD4–183 was purified from E. coli cellpellets using cell disruption, protein solubilization, oxida-tion, Q-Sepharose anion-exchange chromatography, andIA chromatography steps. sCD4–PE40 was purified fromcell pellets using cell disruption, protein solubilization,oxidation, Cu2+-immobilized metal-affinity chromatog-raphy, anion-exchange chromatography, and IA chro-matography steps. The immobilized MAb appeared tobe selective for correctly folded CD4 protein sincesCD4–183 and sCD4–PE40 purified by the IA methodbound the HIV glycoprotein gp120 (HIV gp120) in vitro.The results demonstrate that immobilized MAbs directedagainst conformational epitopes may be used for the rapidpurification of gram amounts of correctly folded proteinfrom mixtures of oxidized E. coli proteins.

5.2.2 Red Blood Cell Membrane

Molecular masses of the proteins contained in red bloodcell membrane (Figure 2) range between 20 and 200 kDa,and consequently, the most common HPLC techniques

used for the purification of these proteins are based onSEC (which may be used in the presence of either ionicor nonionic detergents) and, to a lesser extent, on ion-exchange or hydrophobic interaction chromatography(compatible with nonionic detergents).

We have used an RP C-4 column for the separationof membrane proteins from erythrocyte ghosts usingmobile phases containing acetonitrile and 0.1% TFA. Theerythrocytes were swollen in 5 mM Na2HPO4 isotonicsolution, and after several centrifugations at 3000g thepellet was solubilized in two volumes of pure acetonitrile.Samples were loaded directly on the column without anyother manipulation. Figure 3 shows the chromatogramobtained using a gradient of acetonitrile.

A silica-based C-4 column eluted with TFA–aceto-nitrile is a perfect combination for several polypeptideseparations but was considerably less suitable formembrane protein separation than a resin-based phenylcolumn eluted with the same mobile phase. In ourcase, the separation obtained with Vydac C-4 may beconsidered excellent, better than that reported previouslywith a Nucleosil C-4 column(67) where only one main peakwas observed. In our opinion, the better separation isrelated to the absence of detergent during the extractionand elution run and probably also to the better quality ofresin in the column.

GPGP

Ankyrin Ankyrin

AnkyrinSpectrin dimers

Aldolase

4.24.2

4.2

3

33

Hemichrome

PFK

GAPDH

Aldolase

Hemichrome

PFK

GAPDH4.1 4.1

S

S

Figure 2 A schematic view of the organization of the main red blood cell membrane proteins.



–0.12

0.03

0.18

Abs

orba

nce

0 5 10 15

Time (min)

20 25 30 35 40

Figure 3 Separation of erythrocyte membrane proteinsextracted in pure acetonitrile. Inset shows SDS/PAGEof proteins loaded into the column. Vydac C-4 column(250 × 4.6 mm ID) eluted at 1.0 mL min−1 with an acetonitrilegradient (20–100%) in 0.1% TFA over 60 min.

5.2.3 Red Blood Cell Membrane ProteinsCharacterization Through Preliminary SodiumDodecyl Sulfate Polyacrylamide GelElectrophoresis and High-performance LiquidChromatography/Mass SpectrometryIdentification

Recently, the proteomics workflow including preliminaryseparation steps through gel-based approaches, digestionof bands/spots of interest, and identification by MS hashelped dramatically expand the red blood cell membraneproteome.(68,69)

Packed RBCs (10 mL) were suspended in 50-mL ice-cold 5 mM phosphate buffer, pH 8, and centrifuged(9000g, 20 min, 4 °C). Hemolysate was discarded andthe operation repeated (at least five times) until thesupernatant appeared colorless. Centrifugation was thenincreased to 20 000g, and washing was repeated until theghost membranes appeared yellowish white. Membraneswere stored at −80 °C. Protein extraction has beenperformed with Na2CO3 or EtOH.

In the former case, RBC membranes (10 µL; 80 µgprotein) were resuspended in 1 mL 100 mM Na2CO3 (pH11) and passed five times through a 25-gauge needle,mixed by rotation (30 min, 4 °C), and pelleted (90 min,245 000g), and the supernatant was removed. This processof suspension, rotation, pelleting, and washing wasrepeated twice. The pellet was either digested directlyor treated with EtOH.

In the second protocol of membrane protein extraction,RBC membrane pellets were diluted with four volumesabsolute EtOH and brought to 50 mM sodium acetate,using 2.5 M sodium acetate, pH 5.0. Final pH wasapproximately 7.5. Twenty micrograms of glycogen permililiter of the original sample was added, the suspensionwas mixed at room temperature (RT) for 90 min and

centrifuged (10 min, RT), and the supernatant wasdiscarded.

The whole complex proteomic workflow, includingSDS-PAGE, digestion of separated bands, and capillaryliquid chromatography/MS analysis, yielded identificationof 340 independent protein spots.(68)

5.2.4 From Extensive Basic Studies toward ClinicalApplication

Preliminary 2-D-IEF-SDS-PAGE, trypsin digestion ofspots of interest, and nano-HPLC-ESI-MS have revealedfar more membrane proteins in a single approach.(69)

The erythrocytes were isolated by centrifuging twice at1000g for 10 min. Packed cells were washed three timesin 5 mM phosphate buffer, pH 8.0, containing 0.9% (w/v)NaCl; then, they were centrifuged at 300g for 10 min,at 4 °C. Erythrocytes were lysed with nine volumesof cold 5 mM phosphate buffer, pH 8.0, containing1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride(PMSF). Membranes were collected by centrifugation at17 000g for 20 min at 4 °C and further washed until free ofhemoglobin. To remove lipids, proteins were precipitatedfrom a desired volume of each sample with a coldmix of tri-n-butyl phosphate/acetone/methanol (1:12:1).After incubation at 4 °C for 90 min, the precipitatewas pelleted by centrifugation at 2800g for 20 min at4 °C. Samples were therefore analyzed through 2-D-IEF-PAGE. Spots of interest were carefully excisedfrom the 2-D gel and subjected to in-gel trypsindigestion according to Shevchenko et al.(70), with minormodifications. The gel pieces were swollen in a digestionbuffer containing 50 mM NH4HCO3 and 12.5 ng/µLtrypsin (modified porcine trypsin, sequencing grade,Promega, Madison, WI, USA) in an ice bath. After30 min, the supernatant was removed and discarded;then, 20 µL of 50 mM NH4HCO3 was added to the gelpieces, and digestion was allowed to proceed overnightat 37 °C. The supernatant containing tryptic peptides wasdried by vacuum centrifugation. Before MS analysis, thepeptide mixtures were redissolved in 10 µL of 5% FA(formic acid). Peptide mixtures were separated usinga nanoflow-HPLC system (Ultimate; Switchos; Famos;LC Packings, Amsterdam, The Netherlands). A samplevolume of 10 µL was loaded by the autosampler ontoa homemade 2-cm fused silica precolumn (75 µm ID;375 µm o.d.; Resprosil C18-AQ, 3 µm (Ammerbuch-Entringen, DE, Germany)) at a flow rate of 2 µL min−1.Sequential elution of peptides was accomplished usinga flow rate of 200 nL min−1 and a linear gradient fromsolution A (2% acetonitrile and 0.1% FA) to 50% ofsolution B (98% acetonitrile and 0.1% FA) in 40 min



over the precolumn in-line with a homemade 10- to 15-cm resolving column (75 µm ID; 375 µm o.d.; ResprosilC18-AQ, 3 µm (Ammerbuch-Entringen, Germany)).

Peptides were eluted directly into a high-capacityion trap (model HCTplus Bruker-Daltonik, Germany).Capillary voltage was 1.5–2 kV, and a dry gas flow rate of10 L min−1 was used with a temperature of 200 °C. Thescan range used was from 300 to 1800 m/z.

Although time consuming, this workflow allowedindividuation of protein fragments and aggregatesemerging upon prolonged storage of erythrocyte concen-trates under blood-banking conditions. These evidencesrevealed that either structural or functional proteins sufferfrom irreversible damage as storage progresses. Amongthese proteins, notable was the presence of band 3, bands4.1 and 4.2, glyceraldheyde-3-phosphate dehydrogenase,and ankyrin, which are either structurally or functionallyinvolved in erythrocyte survival in vitro and in vivo.

5.2.5 Separation of Photosynthetic Proteins from theThylakoid Membrane of Chloroplasts

In this section, we present the results of a study performedto develop a rapid and straightforward HPLC method toresolve and identify the protein components of PSI andPSII. These two large complexes, of at least 40 proteins,are present in the thylakoid membrane of chloroplasts(Figure 4) and have the main role of capturing the lightand giving the necessary energy to transfer electrons fromwater to nicotinamide adenine dinucleotide phosphate(NADP) via the two photosystems, PSI and PSII, and anumber of electron carriers.

PSII is embedded in the thylakoid membrane andcontains a reaction center (core) of 21 large and smallerproteins, surrounded by a specific light-harvesting system,which is the major protein–chlorophyll a–b complex(light-harvesting complex of photosystem II (LHCII))and minor antennas called CP29, CP26, and CP24.(72) Incontrast, structural information on PSI at the atomic levelis lacking.

The proteins associated with PSI and PSII have beentraditionally resolved by SDS/PAGE into several closelyrelated hydrophobic membrane proteins, typically in therange of 21–40 kDa.(73) The uncertainty of the numberof proteins in the complex is mainly due to the differentprocedures and detergents used to solubilize PSI and PSIIfrom the thylakoid membranes and the different methodsemployed in isolating and characterizing the individualmembrane proteins in the complex. The antenna proteinsshow amino acid compositions similar in length andsequence,(74) and therefore, the characterization of theseproteins is performed by tedious electrophoresis andimmunoblotting, which require run times of 20–30 h.(75)

Traditional approaches by SDS/PAGE are not onlycumbersome but also rather ineffective for evaluatingdifferences in the relative quantity of each LHCII compo-nent. We have set up a high-performance separationtechnique for the resolution of thylakoid membraneproteins, employing an RP Vydac Protein C-4 columncontaining 5-µm pores.(76)

5.2.5.1 Preparation of Photosystem I and Photosystem IIIn Figure 5 are summarized the main steps to separatethe PSI and PSII present in the thylakoid membrane.

hν

Stroma

MembraneP680

III

CH0

IVII

I

aε g δ

bdEm

βα

β

ATP

αβ

α

3H−

NADP+ADP + Pi

Carbon fixing reactions

FdEe-

KJHCGD

MIL F F

Fx A− AA−

P700

PCNe−

F

B

Lhca1-4

Lhca1-4

b6 FeS IV

Q

cyt b

cyt b

FeSe−

e−

e−

QH−

H−

H−

H−

Fe

PC

PQ−

pool

PQH2

H−H−

Lhcb 1+2+3

Lhcb 4 Lhcb 5 Lhcb 6

J K W I C(CP43) B(CP47)L

S NHM X

QBA

(D1)

QA

(D2)D

PheE F

Mn MnMn Mn

Lumen

H2O

(CP43) Q R

P O

(CP47)T

Y−Y−cytf

e−

O2+

PSII Cyt b6f PSI ATP synthase

hn

oscpCF1

12

12

14

Figure 4 Organization of proteins contained in the thylakoid membrane and involved in photosynthesis.(71) (Reproduced bypermission of Professor J. Barber, Imperial College, London.)



Leaf

Freezing at −80 °C and homogenizationCentrifugation at 1400g for 10 min

Chloroplast

Centrifugation at 10 000g for 10 min

Thylakoids

Solubilization in 1.5% β-DM for 30 minIon exchange by DEAE +Size exclusion by ultragel AcA34(36)

Supernatant PSI + PSII

Concentrated and solubilized in1% β-DM for 20 minUltracentrifugation at 28 000 rpm for42 h in 0–1M sucrose gradient

Solubilization in 1% β-DM for20 minUltracentrifugation at 39 000 rpmfor 18 h in 0–1M sucrose gradient

Pellet PSII

Ultracentrifugation at 40 000g for30 min

PSI

PSI

PSII

41 000 rpmfor 24 h

1

2

3

4

5

1

2

3

4

5

Figure 5 Separation scheme for PSII and PSI.

Chloroplasts were isolated from spinach leavesaccording to the method of Berthold et al.(77) with minormodifications. Leaves were powdered in liquid nitrogenand subsequently homogenized in an ice-cold 20 mM

tricine buffer (pH 7.8) containing 0.3 M sucrose and5.0 mM magnesium chloride (buffer B1). The homog-enization was followed by filtration through one layerof Miracloth (Calbiochem, San Diego, CA, USA) and



centrifugation at 4000g for 10 min at 4 °C. Pellets weresuspended in buffer B1 and centrifuged as above. Thesesecond pellets were resuspended in 20 mM tricine buffer(pH 7.8) containing 70 mM sucrose and 5.0 mM magne-sium chloride (buffer B2) and centrifuged at 4500g for10 min. Pellets containing the thylakoid membranes werethen resuspended in 50 mM morpholinoethanesulfonicacid (MES) buffer (pH 6.3) containing 15 mM sodiumchloride and 5 mM magnesium chloride (buffer B3) at2.0 mg mL−1 chlorophyll for 15 min after adding TritonX-100 at a final ratio of 20 mg mg−1 chlorophyll. Theconcentration of chlorophyll was determined accordingto the method described by Porra et al.(78) The incubationwas terminated by centrifugation at 40 000g for 30 min at4 °C. This pellet containing the PSII complex and corre-sponding to the BBY preparation described by Bertholdet al.(77) was resuspended in buffer B3 containing 20%(v/v) glycerol and stored at −80 °C.

5.2.5.2 Subfractionation of Photosystem II by SucroseGradient Ultracentrifugation Once isolated fromthylakoid membranes, PSII membranes were subjected tosucrose gradient ultracentrifugation in order to isolate theprotein components of the minor antenna system (band 2)from the major (band 3) and also from the reaction-centercomplexes (bands 4–5). To this end, PSII membraneswere pelleted by centrifugation at 10 000g for 5.0 minat 4 °C, suspended in buffer B3 at 1.0 mg mL−1 chloro-phyll, and then solubilized by adding 1% (w/v) β-DM.Unsolubilized material was removed by centrifugation at10 000g for 10 min. The supernatant was rapidly loadedon to a 0.1–1.0 M sucrose gradient containing buffer B3and 5.0 mM β-DM. The gradient was then spun on aKontron (Milan, Italy) Centricon T-1080 ultracentrifugeequipped with a TST 41.14 rotor at 39 000 rpm for 18 hat 4 °C. Green bands were harvested with a syringe. TheSDS/PAGE analysis of these green bands revealed thatband 3 contained essentially LHCII proteins, as reportedpreviously by Bassi and Dainese.(79) The purified materialwas subsequently used for the HPLC study.

5.2.5.3 Separation of Photosystem II Proteins byReversed-phase High-performance Liquid Chromato-graphy In this study,(80) we searched for the condi-tions to resolve the protein components of the majorand the minor antenna system of PSII either as thecomplex isolated by sucrose gradient ultracentrifugationor as assembled in the grana membrane (BBY particles),using an analytical (250 × 4.6 mm ID) or a semiprepar-ative (250 × 10 mm ID) sized column, both packed withthe same 5-µm spherical Vydac C-4 stationary phase.The use of a semipreparative sized column was needed inorder to obtain a sufficient amount of purified polypeptidein order to perform peak identification by SDS/PAGE,

immunoblotting, and amino acid microsequencing. Theoptimum separation of the protein components of thePSII antenna system was obtained by the followingprocedure.

The Vydac C-4 columns were preequilibrated with 38%(v/v) aqueous acetonitrile solution containing 0.1% (v/v)TFA, and samples were eluted using either gradient Ior II, depending on the HPLC unit employed for theseparation. Gradient I consisted of a first linear gradientfrom 38% to 55.4% (v/v) acetonitrile in 22 min, followedby 3 min of isocratic elution with the eluent containing55.4% acetonitrile, a second gradient segment from 55.4%to 61.8% (v/v) acetonitrile in 8 min, and then a thirdgradient segment from 61.8% to 95% acetonitrile in1 min. Gradient II consisted of a first linear gradient from38.0% to 61.8% (v/v) acetonitrile in 40 min, followedby a second gradient segment from 61.8% to 95% (v/v)acetonitrile in 1 min. For both gradients, the last gradientsegment up to 95% acetonitrile was used for washing outhydrophobic contaminants of the PSII antenna systemfrom the column. Gradient I was used to elute eitherthe analytical or the semipreparative column. The flowrate was 1.0 mL min−1 with the analytical column and4.7 mL min−1 with the semipreparative column. Theseconditions were selected in order to maintain the samegradient shape with both columns by keeping the ratio ofthe gradient volume to the column volume constant.

The chromatogram displayed in Figure 6(a) shows thatthe material harvested from the second band of thesucrose gradient ultracentrifugation was resolved intoeight main peaks and contained a mixture of the proteincomponents of both the major and the minor PSII antennasystems. Five of these peaks, with corresponding retentiontimes, were also obtained on separating, under the sameconditions, the material harvested from the third bandof the sucrose gradient ultracentrifugation (Figure 6b),which essentially contained the protein components ofthe major PSII antenna system. From these data, itcan be inferred that the peaks labeled 1, 2, 5, 6, and7 correspond to components present in both the sucrosebands 2 and 3, whereas peaks 3, 4, and 8 are essentiallydue to the component present only in the sucrose band2, which can be tentatively identified with the minorantenna complexes CP29, CP26, and CP24.(80) Figure 6(c)shows the chromatogram of BBY injected on the columndirectly, avoiding the separation step by sucrose gradientultracentrifugation. It can be observed that on injectionof BBY directly, the protein components of the PSIImajor and minor antenna system are well resolvedwithout interference from the other protein componentsof PSII. Therefore, the use of the crude PSII membranepreparation does not affect the resolution and retentiontimes, while new and smaller peaks appear at longerelution times. The new peaks observed in Figure 6(c)



−0.005

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10 20

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Abs

orba

nce

0.58

Figure 6 RP-HPLC separation of spinach chlorophyll a/bbinding proteins contained in (a) band 2 and (b) band 3 ofspinach BBY fractionated by sucrose gradient ultracentrifuga-tion and (c) in whole BBY, directly injected into the column.Column, Vydac Protein C-4 (250 × 10 mm ID), eluted with anincreasing acetonitrile concentration gradient as described inthe text. Flow rate, 4.7 mL min−1; detection at 214 nm.

represent the core proteins: being more hydrophobic,they tend to elute at higher concentrations of acetonitrile(data not shown).

In order to assign a name to each HPLC peak ofthe well-known antenna proteins, SDS/PAGE employingdifferent buffer systems and either Coomassie brilliantblue or silver staining followed by immunoblottingdetection with antisera directed to the individualantenna proteins were performed, in addition to aminoacid microsequence analysis of the material collectedthroughout the chromatogram.

In the case of SDS/PAGE and immunoblotting, eachfraction collected from the semipreparative chromato-graphic separation lyophilized were dissolved in 120 mMtris(hydroxymethyl)aminomethane (TRIS)-HCl buffer(pH 8.45), containing 120 mM dithiothreitol (DTT),5 M urea, and 4% (w/v) SDS, and then analyzed

by SDS/PAGE, according to the method reported byShagger and von Jagow(81) (Figure 7a).

Following electrophoresis, the gels were either silverstained or transferred to nitrocellulose. Replicates wereassayed with antisera directed to LHCII, CP29, CP26,and CP24(82) (Figure 7b–d). Because of the high degreeof homology shared by all the antenna polypeptides,it was essential for identification of the fractions totake into account both the immunoreactivity and theelectrophoretic mobility of the SDS/PAGE bands. InTRIS/Tricine electrophoresis, antenna proteins migratein the following order of increasing mobility: CP29 >

CP26 > LHCII > CP24 and within LHCII, Lhcb1 >

Lhcb2 > Lhcb3.(83) Accordingly, the slower-migratingband was detected by the anti-CP29 in peak 4 (Figure 7b),while peak 8, containing a slightly more mobile band,was recognized by anti-CP26 (Figure 7c). The anti-CP24detected several bands (Figure 7d), but the strongestsignal, with respect to the intensity of the band in thesilver-stained gel, was obtained with the most mobileband in peak 3. Therefore, peaks 3, 4, and 8 seem tocontain CP24, CP29, and CP26, respectively. The higherhydrophobicity of CP26 agrees with the longer elutiontime required.

Thus, according to the migration order of increasingmobility reported above, the fastest migrating band wasrecognized as Lhcb3, the second fastest migrating bandas Lhcb2, and the two newly resolved bands as Lhcb1components of LHCII.

From the above data, we conclude that peak 1 doesnot contain protein components; peak 2, the Lhcb2component of LHCII; peak 3, the CP24 (Lhcb6); peak 4,CP29 (Lhcb4); peaks 5 and 6, two Lhcb1 components ofLHCII; peak 7, Lhcb3; and peak 8, CP26 (Lhcb5).

In order to support the identification previouslyassigned, the protein contained in each HPLC peak wassubjected to amino acid microsequencing and comparedwith amino acid sequences reported in the literature forother species (data not shown). Good agreement with theabove assignment was found.

Finally, the assignment of each peak resolved by RP-HPLC performed by electrophoresis, immunoblotting,and amino acid sequencing is corroborated from thevalues of molecular masses determined by the combineduse of microbore HPLC coupled on-line with a massspectrometer(84) equipped with an electrospray ion source(Section 6).

From the results presented, it can be inferred that themajor antenna system of PSII isolated from spinach leavescontains two different Lhcb1 proteins that can be resolvedby the RP-HPLC system employing a 250-mm long VydacC-4 column eluted with a multistep acetonitrile gradientand by the high-resolution TRIS/Tricine SDS/PAGEsystem in gels of 15 cm length (data not shown). On



66(kDa)MW

MW 1 2 4 5 6 7 8 C3

4536292420

14

(a) (b)

(c) (d)

Figure 7 Gel electrophoresis and immunoblotting of each protein collected throughout the chromatographic run reported in (a).Lanes correspond to the peak labels in (a); the last lane (c) represents the LHCII from Zea mais used as a control. (a) SDS/PAGEof uniform polyacrylamide concentration (13%) with TRIS–Tricine system of each collected peak, which were dried and thensolubilized in 4% SDS, 120 mM DTT, and 120 mM TRIS-HCl (pH 8.45), containing 5 M urea. (b) Immunoblotting of the gel reportedin (a) detected by using antisera directed to CP29. (c) Immunoblotting of the gel reported in (a) detected by using antisera directedto CP26. (d) Immunoblotting of the gel reported in (a) detected by using antisera directed to CP24. (Reprinted from PhotosynthesesResearch 61 (3), 1999, 281–290, Zolla et al. Copyright (1999), with kind permission from Springer Science and Business Media.)

the other hand, the small microsequence performed doesnot reveal any amino acid differences, and the molecularmass measured by MS indicates that the difference isin the order of 60–80 kDa. Further experiments and acomplete amino acid sequence are necessary to explainthese different subpopulations of type I. Nevertheless,the existence of more type I LHCII is in accordance withmolecular genetic data reported in the literature showingthat higher plants have several Lhcb1 genes encodingdifferent Lhcb1 proteins for each species.(75) However, itis important to note that the resolution by SDS/PAGE oftwo Lhcb1 proteins usually requires special experimentalconditions such as the use of polyclonal and monospecificantibodies,(85) dedicated electrolyte solutions, and a gelof extended length,(86) whereas using HPLC the twodifferent subpopulations may be easily separated.

The RP-HPLC method reported here, in addition tobeing rapid, simple, and precise, has proven to be effectiveat detecting differences in the protein components ofLHCII isolated from different plants that might not beevidenced by denaturating SDS/PAGE, as in the case

of type I of spinach. This knowledge is expected to shedlight on the composition and supramolecular organizationof LHCII and may increase the understanding ofthe molecular mechanisms underlying the physiologicaladaptations of higher plants to environmental conditions.

5.2.6 Separation of Proteins Present in Photosystem I

PSI prepared from the thermophilic cyanobacteriumSynechococcus elongatus has been crystallized and thestructure determined at a resolution of 6 A.(87) Thedetermination of the structure of higher-plant PSI hasbeen less successful, most likely because it is a muchlarger complex than its cyanobacterial counterpart. Thecyanobacterial PSI complex consists of 11 subunits.(88) Inthe case of higher plants, although a lot of PSI genes havebeen identified and sequenced, there is little informationabout the actual proteins, and the relationship betweengene and protein expression. This is related to the factthat the molecular masses of most of these proteins range



between 4 and 25 kDa, and consequently, they are notwell separable by SDS/PAGE.

5.2.6.1 Isolation of Photosystem I by Sucrose GradientUltracentrifugation In the first phase of experiments,supernatants were concentrated by ultrafiltration andthen solubilized by β-DM at a final concentration of1%. After stirring for 20 min at 4 °C, the sample wascentrifuged for 10 min at 40 000g, and 6-mL aliquotsof the supernatant were loaded on 0.1–1 M sucrosegradients (35 mL) layered over 2 mL of 2 M sucrose,containing 5 mM Tricine (pH 7.8) and 0.03% β-DM. Aftercentrifugation for 42 h at 28 000 rpm in an SW28 rotor(Beckman) at 4 °C, four green bands were distinguishable.The bands were collected and analyzed by SDS/PAGE.(89)

The lowermost band, containing PSI-200, was dilutedin 5 mM Tricine (pH 7.8) and centrifuged for 3 h at70 000 rpm in an 80 Ti rotor (Beckman). The pellet wasresuspended in 5 mM Tricine (pH 7.8) and 50 mM sorbitol,frozen in liquid nitrogen, and stored at −80 °C.

5.2.6.2 Purification of Photosystem I Core and Light-harvesting Complex of Photosystem I The pellet fromPSI-200 preparation was resuspended at 0.3 mg mL−1

in distilled water and solubilized by 1% β-DM and0.5% Zwittergent-16. After stirring for 20 min at 4 °C,the sample was rapidly frozen in liquid nitrogen andslowly thawed to improve the detachment between thePSI core and the light-harvesting complex of photosystemI (LHCI). Samples (1.5-mL aliquots) were loaded on a12-mL 0.1–1 M sucrose gradient, also containing 5 mMTricine (pH 7.8) and 0.03% β-DM. After centrifugationfor 24 h at 4 °C at 41 000 rpm in an SW41 rotor(Beckman), five green bands were obtained. The bandswere analyzed by SDS/PAGE.(89) The second band fromthe top contained all the LHCI polypeptides, and thethird band contained the PSI core particles. The fractionswere frozen in liquid nitrogen and stored at −80 °C.

5.2.6.3 Separation of Photosystem I Proteins by Reversed-phase High-performance Liquid ChromatographyThe complete resolution of the protein componentsof the PSI was performed by RP-HPLC as for PSII.The experiments were carried out by performingchromatography of all proteins of PSI-200 and thenseparately for the antenna and core proteins, whichhad been previously isolated by sucrose gradientultracentrifugation.

Figure 8(a–c) compares the chromatograms obtainedupon injection of (a) isolated antenna proteins, (b) thecore proteins, and (c) the total PSI-200.(89)

Different gradients were used to find the optimumexperimental conditions to resolve the protein compo-nents of the antenna and core complex. In the case

0

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PsaE PsaF PsaL,F,GPsaA+B

PsaD

PsaH.C

Lhca1

Lhca4Lhca2

(c)

3

Figure 8 RP-HPLC separation of (a) spinach LHCI proteinscontained in band 2 and (b) core proteins in band 3 of PSI-200fractionated by sucrose gradient ultracentrifugation and (c) inwhole PSI-200. Column, Vydac Protein C-4 (250 × 10 mm ID),eluted with an increasing acetonitrile concentration gradientas described in the text. Flow rate, 4.7 mL min−1; detection at214 nm.

of PSI-200, the optimum separation of all proteins wasobtained with a multistep gradient. Good complemen-tarity between the three chromatograms is observed,indicating that the presence of all proteins in the PSI-200 system does not affect the elution time of eachprotein. The material harvested from the second bandof sucrose gradient ultracentrifugation is resolved intofive main peaks (Figure 8a). Previous analyses withSDS/PAGE showed that it contained a mixture of theprotein components of the PSI antenna system. Most ofthe chromatographic peaks are symmetrical, but the lastone at 43.4 min is broad, indicating the presence of morethan one protein. In contrast, the material harvested fromthe third band of the sucrose gradient ultracentrifugation,containing essentially the core protein components of



PSI, shows a main peak at a long elution time of 61 minand numerous smaller peaks. The main peak is broad andelutes at a long time, suggesting the presence of morethan one protein showing high hydrophobicity. Finally, inthe chromatogram for the total PSI-200, five of the samepeaks are present, with retention times correspondingto those observed in Figure 8(a), which are the mostabundant separated proteins and allow one to hypothe-size that they represent the antenna complex, althoughonly four proteins are expected. Moreover, the mainpeak observed in the core complex (Figure 8b) is alsoobserved in Figure 8(c), which may be hypothesized tocontain psaA and psaB, and other smaller core peaks areunderestimated when all proteins are present.

Once the optimized conditions to obtain a betterseparation of each component had been found, weattempted the identification of each protein contained ineach peak by coupling on-line HPLC with MS (Section 5).The mass values measured by ESI-MS correspondedto the molecular mass expected on the basis of theDNA sequence, allowing the assignment of each peakas reported in Figure 8(c).(89) These results corroboratethe utility of coupling an HPLC/MS on-line with anelectrospray ion source.

Finally, the separation of both PSI and PSII may beachieved by direct injection of thylakoid membrane intothe column and elution with a multistep gradient. All theprotein components of PSII can be well resolved withoutinterference from the other protein components of PSI,indicating that the use of the crude thylakoid membranepreparation does not affect the resolution and retentiontimes of each protein.

5.2.7 Comparison of Monolithic and Packed CapillaryColumns in the Separation of Photosystem IComponents

An analytical study of the loading capacity of themonolithic stationary phase with that of a beaded, totallyporous, PS/DVB stationary phase (Vydac 259VHM,5 µm, 300 A) revealed that the loading capacities ofthe monolithic and the beaded stationary phases areabout the same for large proteins of Mr > 50 000,whereas the amount of polypeptide of Mr < 15 000 thatcan be loaded onto the porous stationary phase isapproximately 10-fold that of the monolithic one.(90)

The reason for this discrepancy is the difference inaccessibility of the chromatographic surface to analytesof different molecular size. The surface in the microporesof porous separation media is not accessible to largebiomolecules; thus, the loading capacity decreasesrapidly with increasing molecular mass. Moreover,compared to octyl or octadecyl stationary phases, PS-DVB monolithic stationary phases are known to be mildly

hydrophobic(91,92), which makes them eminently suitablefor the separation of very hydrophobic membraneproteins. In this review,(93) the applicability of the RP-HPLC/ESI-MS method (using both monolithic capillaryand beaded columns) to the analysis of larger proteinsin biological samples in a difficult matrix is demonstratedusing the protein components of PSI of thylakoidmembranes whose molecular masses range between 3500and 80 000. Figure 9 compares the separation of all PSIcomponents performed by capillary monolithic and abeaded silica capillary filled with C4 RP. The separationtime of the monolithic column is almost halved comparedto that expected of the same protein mixture on aconventional, butyl-silica stationary phase with 300-Apores, and the resolution of the peaks is significantlybetter. The peak widths at half-height ranged from 3 to 5s, which demonstrates the high resolving power and speedof analysis with monolithic PS/DVB capillary columns.However, in both cases, mass spectra of high quality wereextracted from the reconstructed ion chromatograms,showing no adduction with detergent. Interestingly, theelution order changed from Lhca1< Lhca3< Lhca4<

Lhca2< PsaF on the silica-based stationary phase toLhca1< Lhca4< Lhca3< PsaF< Lhca2 on the monolithiccolumn, which points to differences in selectivity betweenthe silica-bonded and the polymeric stationary phase.

5.2.8 Reversed-phase High-performance LiquidChromatography/Electrospray Ionization MassSpectrometry of Entire Thylakoid Membranes

In this last section, we present the results of astudy designed to develop a rapid and straightforwardmethod, using HPLC, to resolve and identify the proteincomponents of both the PSI and PSII, starting from theentire thylakoid membrane.

Normally, before loading a complex biological matrixon any HPLC system, membrane proteins must be presep-arated according to their hydrophobic characteristics, andthis is achieved by selective extractions. This pretreat-ment facilitates subsequent separation and provides afirst guideline for the choice of detergent. As a rule, thedetergent chosen for the running buffers is the same onewith which the protein was solubilized. Also, in order toretain the biological activity of proteins, it is preferableto use less denaturing detergents and nonionic detergentsfor solubilization and in the running buffers (the inter-ested reader is referred to the article by Seddon et al.(8)

for further details on the use of detergents for membraneprotein analysis). The presence of detergent in all stepsminimizes the tendency toward association and aggrega-tion as well as the possibility of nonspecif‘ıc interactionswith the support used for chromatographic separation.



0(a)

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C; P

saE

; Psa

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A: P

saB

Lhca

1.1

Lhca

1.2

Lhca

3 Lhca

4Lh

ca2.

1

Lhca

2.2

Figure 9 Comparison of RP analysis of PSI subunits from barley performed by PS-DVB-monolith and C4-beaded column.(a) Column, PS-DVB-monolith, 60 × 0.10 mm ID; mobile phase, (solvent phase A) 0.050% trifluoroacetic acid in water, (solventphase B) 0.050% trifluoroacetic acid in acetonitrile; linear gradient, 39–49% B in 20 min, 46–55% B in 10 min, and 55–99% B in15 min; flow rate, 2.0 µL min−1; and temperature, 60 °C. (b) C4-beaded capillary, 150 × 0.18 ID; mobile phase and gradient as (a);flow rate, 2.0 µL min−1; and temperature, 25 °C.

The first consideration in defining an HPLC methoddevelopment strategy is to establish if we need proteinswith their biological properties intact or if we simplyneed to characterize them. There are many factors to betaken into consideration during the separation develop-ment process in order to select appropriate conditionsand an appropriate detergent, under which artifac-tual denaturation is minimized. Factors arising fromthe presence of detergent must be taken into accountalong with the possibility of nonspecifıc interactionswith the support used for chromatographic separa-tion.

Because of the high complexity of the photosyntheticapparatus, liquid extraction followed by sucrose gradientultracentrifugation was chosen as the sample preparationand prefractionation method.(93) The membrane proteinsheld in the suspended thylakoid membranes at a finalconcentration of 1 mg chlorophyll per mililiter weresolubilized by adding DM (dodecil maltoside) to a finalconcentration of 1% (w/v). After stirring for 20 min at4 °C, the sample was centrifuged for 10 min at 20 000g toeliminate insoluble material. One-mililiter aliquots of thesupernatant were loaded onto 12-mL ultracentrifugationtubes containing 0.5 mol L−1 sucrose, 5 mmol L−1 Tricine,

pH 7.8, and 0.030% DM. After centrifugation (rotor TST41.14) for 42 h at 140592 × g at 4 °C, four green bandswere distinguishable.

The green bands were harvested with a syringe. SDS-PAGE analysis of the fractions from spinach revealed thatfraction 1 contained a mixture of the protein componentsof the minor and monomeric major PSII antennasystems, whereas fraction 2 essentially contained theprotein components of the trimeric major PSII antennasystem, as previously reported. Fraction 3 contained thereaction-center complex of PSII, and finally, fraction4 contained the subunits of PSI.(93) The bands weresubsequently analyzed by RP-HPLC/ESI-MS using PS-DVB-based monolithic capillary columns. Monolithiccapillary columns (60 × 0.20 mm ID and 60 × 0.10 mmID) were used for protein separation. ESI-MS wasperformed on a quadrupole ion trap mass equipped witha triaxial electrospray ion source. An electrospray voltageof 1.6–2.0 kV and a nitrogen sheath gas flow were usedfor analysis with pneumatically assisted ESI. Fine tuningfor ESI-MS of proteins was accomplished by infusion of a6.9 pmol µL−1 solution of carbonic anhydrase in 0.050%aqueous TFA solution containing 20% acetonitrile (v/v)at a flow rate of 3.0 µL min−1.



The analytical method was shown to be very flexibleand allowed the antenna proteins to be identified, aswell as most of the proteins of the reaction center fromPSI and PSII in various plant species, requiring few RP-HPLC/ESI-MS analyses and only minor adaptations inthe acetonitrile gradients in 0.05% aqueous TFA. Themembrane proteins, ranging in molecular mass from 4196(I protein) to more than 80 000 (PSI A/B), as well asisoforms, were identified on the basis of their intactmolecular masses and comparison with molecular massesdeduced from known DNA or protein sequences.

Figure 10 depicts the RP-HPLC/ESI-MS analysis ofPSII reaction-center subunits contained in fraction 3.Chromatography at elevated temperatures facilitated

00

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b H

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Aps

b C

psb

D

psb

L

30

Time (min)

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Figure 10 RP-HPLC/ESI-MS analysis of PSII core proteinsof spinach performed at different temperatures. Column,PS-DVB-monolith, 60 × 0.20 mm ID; mobile phase, (solventphase A) 0.050% trifluoroacetic acid in water, (solvent phaseB) 0.050% trifluoroacetic acid in acetonitrile; linear gradient,0–50% B in 30 min and 50–62% B in 10 min; flow rate, 2.5 µLmin−1; temperature, (a) 65 °C, (b) 74 °C, and (c) 78 °C; detection,ESI-MS, scan, and 600–2000 amu; electrospray voltage, 1.6 kV;sheath gas, nitrogen; and sample, fraction 3 of spinach.

elution of the membrane proteins as very sharppeaks, although a relatively shallow gradient wasapplied for elution. Most of the proteins of thereaction center were well separated at 65 °C butthe large proteins psb C and psb D co-eluted atthis temperature (Figure 10a). Nevertheless, a shoulderappeared in the peak eluting around 35 min at 74 °C,and the proteins were almost separated to baselineat 78 °C (Figure 10c). The molecular masses of thesubunits obtained upon deconvolution of the raw massspectra that were extracted from the reconstructedtotal ion current (TIC) chromatogram were in excellentcorrespondence with the molecular masses expectedfrom the DNA sequence (including posttranslationalmodifications), wherever relevant. Thus, high- andlow-molecular-mass PSII reaction-center subunits weresuccessfully analyzed by ESI-MS following off-line RP-HPLC fractionation.(94) Nevertheless, molecular massdetermination was relatively inaccurate because of co-eluting compounds, especially in the high mass range.(95)

Moreover, some of the high-molecular-mass subunits,such as psb C or psb D, could not be detected atall.(95) Because of the high hydrophobicity of the psbA and psb D subunits, they were difficult to elutefrom an octyl-silica column (Aquapore RP-300) with 1-propanol/5% acetic acid.(95) Because of the high chemicalstability of the polymeric stationary phase, analysiswith PS-DVB monoliths can be performed routinelyat elevated temperatures, which significantly enhanceschromatographic performance and elution characteristicsof proteins.

High-quality mass spectra made it possible to identifyand quantify the nonphosphorylated and phosphorylatedreaction-center subunits D1, D2, and CP43 of PSII,containing five to seven membrane-spanning α-helix. Asemiquantitative estimation of protein phosphorylationwas made by comparison of the relative signal intensitiesof the phosphorylated and nonphosphorylated proteinmass peaks. psb A and psb D showed quite similarpercentages of phosphorylation, namely, 42% and 39%,respectively, whereas psb C was predominantly presentin the phosphorylated form (69%).(95) The measuredmolecular mass of psb H (7698) indicated the presence ofone phosphorylation and one oxidation.

Thus, because of its great flexibility and suitability forproteins having a very wide range of molecular massesand hydrophobicities, the method is generally applicableto the analysis of complex mixtures of membraneproteins. Moreover, intact mass measurements by ESI-MS provide mass accuracy, often exceeding 0.01%(100 ppm), and resolution sufficient to observe the firstoxidation adduct (16 Da) or phosphorylation (79 Da),providing an attractive alternative technique to monitorthe subtle changes that often accompany physiological



adaptations. This presents a new facet to intact massproteomics as a potent tool in proteomics.

5.3 Separation of Lipoproteins

5.3.1 Preparation of Lipoprotein Fractions

Plasma or serum can be used for lipoprotein analysis.However, plasma is preferred since it can be kept cold inorder to slow most enzymatic processes that degradelipoproteins. The following procedure is commonlyused to prepare lipoproteins.(96) Human blood (Ca20–30 mL) was collected from normolipidemic malesby venipuncture after 12–16 h of fasting. The bloodwas allowed to stand for 2–3 h at room temperatureuntil agglutination was complete. Plasma was withdrawnafter centrifugation at 1000g at 15 °C for 15 min. Theplasma density was adjusted to 1.225 g mL−1 by addingsolid potassium bromide (0.3517 g KBr per milliliter ofplasma). Plasma (1.225 g mL−1, Ca 3–5 mL) was thenplaced in ultracentrifuge tubes, which were centrifugedin a swinging-bucket rotor at 200 000g at 10 °C for 40 h.The lipoprotein fraction prepared by this procedure didnot contain serum proteins, except for a small amount ofalbumin. The lipoprotein fraction in the KBr solution wasdialyzed against 0.154 M sodium chloride solution.

5.3.2 Preparation of Individual High-densityLipoprotein, Low-density Lipoprotein, and VeryLow-density Lipoprotein Fractions

Many separations subfractionate the lipoprotein fractionof plasma. Hence, each main class of lipoproteins maybe collected by ultracentrifugation using a multiplediscontinuous density gradient. For this purpose, humanblood was collected from fasting normolipidemic healthymales in tubes containing 0.15% EDTA. The plasmawas separated by centrifugation at 700 rpm at 7 °C for20 min. A discontinuous NaCl–KBr density gradient(total volume 18.5 mL) was formed by adjusting thedensity of plasma to 1.30 g mL−1 with solid KBrand sequentially layering on the adjusted plasma salt(NaCl–KBr) solutions with densities of 1.240, 1.063,1.019, and 1.006 g mL−1 and 0.5 mL of distilled water.Tubes loaded with the discontinuous density gradientwere placed in a vertical rotor and centrifuged at 313 500g

at 7 °C for 80 min.

5.3.3 Separation of Particles and Subfractions In analternative to ultracentrifuge separation, the GPC modeof HPLC is the most commonly used method toseparate particles such as HDL, VHDL, and LDL. Twocommercial columns are commonly used: Superose 6(from Pharmacia) and TSK type. Rigid, fast-flow agarose

gels, such as Superose 6, are able to accept large samplevolumes (up to 5 mL) and preferred for the preparationof larger quantities of plasma lipoproteins. Furthermore,multiple automated separations of lipoproteins fromwhole plasma may be performed with a preparative-gradeSuperose 6 column. Thus, 2 mL of plasma was loaded andeluted with 0.9% sodium chloride and 2 mM sodiumphosphate (pH 7.4). The eluent also contains 0.02%or 1% sodium azide for serum or plasma separation,respectively. VLDL, LDL, and HDL can be separatedin a single run. A single separation can be completed inabout 160 min. Hence, the system can be used to analyzeup to six different plasma samples (2 mL per sample)overnight.

Fast-flow separation of plasma is also used, wherelipoproteins are detected on-line at 500 nm after post-column derivatization with an enzymatic cholesterolreagent (CHOD-PAP, Boehringer Mannheim). Theeluting lipoproteins and the cholesterol reagent are mixedin a chamber attached to the column outlet. The mixtureis then passed through a reaction capillary and detected.Complete separation of lipoprotein fractions is usuallyachieved in<80 min. The chromatographic profiles ofnormal and several types of hyperlipoproteinemic (HLP)serum samples are illustrated in Figure 11(a–d). Thethree peaks correspond to, in order, VLDL, LDL, andHDL.(97)

Separation of lipoproteins by HPLC on TSK-typegel permeation columns is faster than on a Superose6 column, but smaller sample volumes (<300 µL) maybe loaded. Furthermore, serum instead of plasma ispreferred for TSK-gel HPLC of lipoproteins. Withplasma, there is a danger of fibrin forming during theanalysis. A further disadvantage of the TSK methodis that this type of column is generally more proneto clogging than a Superose 6 column. Adsorption oflipoproteins on the support has also been noted by someworkers. It was found that a combination of G4000 SWand G3000 SW eluted with 0.15 M NaCl (pH 7) was best atseparating serum lipoproteins into VLDL, LDL, HDL2,and HDL3.(98) Separation by a TSK column allows thedetermination of lipids using a combination of two TSKcolumns and on-line enzymatic reaction. The system usedtwo detectors. The first was placed immediately after thecolumn and monitored protein absorbance at 280 nm. Thesecond detector was placed after the enzymatic reactorand detected lipid absorbance at 500–600 nm.(98)

Separation of large amounts of LDLs and VLDLsfrom human serum may be performed by using ahydroxyapatite Bio-Gel HTP DNA grade column (25 ×1.0 cm ID) by one or four stepwise elutions.(36) In thelatter case, a 2-mL volume of human serum is loadedon the column and eluted with a discontinuous gradientof 75, 200, 300, and 650 mM potassium phosphate buffer



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Figure 11 Chromatographic profile of normal and HLP sera. Conditions: 300-mm Superose 6 column eluted with 100 mMNa2HPO4 and 200 mM NaCl (pH 7.4) at 300 mL min−1. (a) Normolipidemic sample; (b) type IIa HLP; (c) type III HLP; and(d) type IV HLP. Peaks from left to right: VLDL, LDL, and HDL. (Reproduced by permission of Clinical Chemistry from W. Marz,R. Skeimer, H. Scharnagi, U.B. Seiffert, W. Gross, Clin. Chem., 39(11), 2276–2281 (1993).)

200 nm

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75 mM KPi (pH 7.4)

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Serumproducts

Figure 12 Stepwise elution profile of human serum lipoproteins. Column, Bio-Gel HTP DNA grade 25 × 1.0 cm ID; eluents, 25,200, 300, and 650 mM potassium phosphate buffer (KPi) (pH 7.4); flowrate, 12.0 mL h−1; and sample, 2.0 mL of human serum.(Reproduced by permission of Elsevier Science from Y. Shibusawa, J. Chromatogr. B, 699, 419–437 (1997).)

at pH 7.4. Figure 12 shows the elution profile of humanserum obtained with a hydroxyapatite column.

Four peaks are detected, the first containing HDLs andserum proteins; the second, the serum proteins, eluted at200 mM; the third, mainly LDLs, eluted at 300 mM; andthe fourth, VLDLs, eluted at 650 mM.

The whole lipoprotein fraction (d < 1.21) can befurther subfractionated into VLDL, LDL, and HDLby GPC.(40) Lipoprotein fractions prepared by

ultracentrifugation are often used for this furtherchromatographic separation. In this way, the proce-dure is both analytical and preparative since theisolated fractions contain no contamination from serumproteins.

Subfractionation of HDL particles accomplished byGPC gives rise to subfractions having different sizes(3.81–528 nm) and different amounts of proteins: HDL2a,HDL2b, HDL3b, and HDL3c.(40)



HDL particles may also be subfractionated byheparin affinity chromatography according to the apoEcontent.(42) HDLs rich in apoE were separated into fivesubfractions using an elution buffer with varying Mn2+concentration.(41)

In the case of LDL, charge heterogeneity was demon-strated by ion-exchange chromatography. These typesof separations are important since oxidatively modi-fied LDLs have been associated with atherosclerosis.(91)

LDL particles modified by Cu2+ were analyzed byanion-exchange chromatography using a Mono Q-HR515 column (Pharmacia).(43) Five different oxidativelymodified LDLs were identified. The isolated fractionsexhibited differences in density and lipid content.

5.3.4 Separation of Apolipoproteins and IsomericForms

The growing evidence that apolipoproteins are bettermarkers of CHD than serum cholesterol levels(35) hasincreased the interest in analyzing lipoproteins accordingto the apolipoprotein compositions.

Affinity chromatography, which employs specific andreversible interactions between lipoproteins and ligands,is capable of separating lipoproteins on the basis oftheir apolipoprotein content by using nonspecific bindingand IA. Heparin was successfully immobilized on toglyceryl CPG and used to bind specifically β-lipoproteins(apoB).(98) Serum samples applied to heparin–CPG wereseparated into two fractions. The unretained fractionwas washed through the column with 0.1 M NaCl,and the retained fraction was eluted with 1 M NaCl.Radial immunodiffusion studies confirmed the completeresolution of apoA- and apoB-containing lipoproteins.However, attempts at incorporating the heparin–CPGcolumn into a high-performance affinity chromatographysystem were unsuccessful. Two types of lipoproteins havebeen clearly established as atherogenic, the apoB100-containing LDL and apoB48-containing chylomicronremnants.(99) Human genetic disorders resulting inincreased circulating levels of either of these lipoproteinscause premature atherosclerosis. Experiments werereported to demonstrate that the NH2-terminal regionof apoB binds to heparin affinity gels with an affinityequal to or greater than that of apoB100-containingLDL.(100) In addition, apoB48-containing lipoproteinswere observed to bind to heparin and LDL. Hence, basedon these findings, it was proposed that NH2-terminal apoBcontributes to the atherogenicity of LDL and remnantlipoproteins.

Dextran sulfate is a synthetic analog of heparin, whichbinds preferentially to apoB-containing lipoproteins.Commercial sulfated dextran beads from Sigma consistof sulfated cross-linked dextran and are useful for

rapid (15 min) separation of apoA- and apoB-containinglipoproteins on elution of proteins by NaCl at 80 mM.

IA chromatography provides the highest specificity forseparation and allows the separation of relatively largevolumes (1–10 mL) of whole plasma or lipoproteins.Preparations of both polyclonal antibodies(101) andMAbs(102) to apolipoproteins, produced using purifieddelipidized apolipoproteins, are commonly used asantigens. Antibodies to apolipoproteins coupled tocross-linked dextran (Sephadex) or agarose derivatives(Sepharose) serve as selective ligands for the separationof lipoproteins by IA chromatography.

Separation of apoproteins into their isomers is ofinterest especially for apoA and apoB, which may beperformed by sequential chromatographic stages usingmore IA or affinity chromatography followed by gelpermeation, ion-exchange, or RP techniques. In thefirst procedure, whole plasma is first fractionated intoapoB and apoA lipoproteins by affinity chromatographyon concanavalin A (con A). Further subfractionationproceeds by subsequent IA chromatography using aspecific antibody: anti-apoA-II immunosorbent, whichretains a fraction containing lipoprotein (A-I + A-II) andlipoprotein A-II. Further, separation of this fraction on ananti-apoA-I immunosorber leads to the isolation of threetypes of apoA lipoproteins: lipoprotein A-I, lipoproteinA-II, and lipoprotein (A-I + A-II).(103)

Conventional GPC or ion-exchange chromatographyon soft gel supports can be used for the preparation oflarge quantities (10–50 mg) of pure apolipoproteins,(104)

whereas by HPLC, only small amounts of sample (<5 mg)can be applied to the column but the analysis is completedwithin 60 min. Separation by high-performance GPCis strongly dependent on the completeness of thedelipidation step, which may be performed satisfactorilywith an organic solvent. However, high-performanceGPC separation of HDL apolipoproteins has beenachieved without prior organic solvent delipidation ofthe HDL fraction.(105)

GPC is often used as a preparative technique forisolating apolipoproteins derived from HDL or VLDL,while HDL apolipoproteins, apoA-I, apoA-II, and apoC,can be separated on Sephadex G-200 columns usingTRIS-HCI buffer (pH 8.6) containing 8 M urea.(106)

apoC and apoE from VLDL can be isolated by SepharylS-200 (Pharmacia) chromatography.(106) Separation ofapolipoprotein isoforms can be achieved by either ion-exchange or RP-HPLC.

Apoc isoforms (C-III0, C-III1, and C-III2) can beseparated on Mono Q-HR 5/5 (Pharmacia) anion-exchange columns(107) in less than 30 min using a lineargradient of 0.15 M NaCl in TRIS-HCl containing 6 M urea(pH 8.2). This mode of apolipoprotein separation is basedon differences in isoelectric points. Chromatography



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Figure 13 RP-HPLC separation of delipidated rat HDL(200 mg protein) on a TSK Phenyl-5PW column. Gradientbetween 20 mM H3PO4 in water (pH 2.3) and 20 mM H3PO4in 60% acetonitrile. Flow rate, 1 mL min−1 at 45 °C. Fractionswere analyzed by SDS/PAGE and their lipoprotein contents asassessed by Coomassie brilliant blue staining (bold bars) andby silver staining (normal) are as indicated. (Reproduced bypermission of Elsevier Science from B. Meyer, E. Kecorius,P. Barter, N. Fidge, T. Tetaz, J. Chromatogr. A, 540, 386–391(1991).)

of VLDL and HDL apolipoproteins is performed onanion-exchange columns such as SynChropak AX300(SynChrom IUC) and Mono Q-HR (Pharmacia).(108)

A rapid and highly efficient technique for monitoringheterogeneity in apolipoproteins is the separation onthe basis of differences in hydrophobicity by RP-HPLC.Unfortunately, the small amounts of apolipoprotein(50–100 µg), which may be loaded, render this methodqualitative but highly resolutive. Successful separationsof apoA and apoC were performed on C-18 columns witha gradient of acetonitrile–water in the presence of 0.1%of TFA.(109) Figure 13 shows the separation of humanapolipoproteins A-IV, A-I, and E on a TSK Phenyl-5PWcolumn.(110)

6 CURRENT TRENDS

6.1 Membrane Proteins

Most of the methods described in previous sections,although seemingly outdated, still represent valid, ifnot unique, approaches to delve into the complexity ofmembrane proteins.(111)

6.1.1 Perfusion Chromatography

The advent of perfusion chromatography, which consistsof matrix containing flow-through particles, has furtherincreased the interest in using HPLC systems, especiallyfor membrane proteins.(112) In contrast to conventionalparticles, the new matrix has two classes of pores.

Large ‘throughpores’ allow convective flow through theparticles, quickly carrying sample molecules to short‘diffusive’ pores inside. By reducing the distance andtherefore the time over which molecules diffuse toaccess the particle-binding surface area, the flow ratecan be increased 10- to 100-fold with little or no lossin resolution or capacity. Thus, once a particular modeof chromatography has been selected, it is possible toperform it by using perfusion particles. In general, theperfusion chromatography allows

• routine achievement of high-resolution laboratory-scale separations in 30 s–3 min;

• rapid and systematic determination of the bestseparation method for optimum purity;

• a significant reduction in the time to process large-volume samples;

• improved yields and recoveries of biological activitythrough faster processing.

6.1.2 High-performance Liquid ChromatographyCoupled On-line with Mass Spectrometry

Further developments in HPLC system have beenachieved by coupling the outlet on-line with otherinstrumentation. The introduction of electrospray ionsources that are coupled with quadrupole mass filters hasproduced a mass spectrometer that is easily compatiblewith HPLC and CE, and therefore, analysis andmeasurement of biological samples at a reasonable costmay be performed.(113,114) Since most MS ion sourcesrequire lower flow rates than common chromatographiclevels, a split of the HPLC eluent stream must beaccomplished. Such systems are highly advantageousin that they facilitate the simultaneous separation ofcomplex mixtures along with ultraviolet (UV) adsorptionand mass detection.

With regard to the separation of thylakoid proteinsof the PSII, RP-HPLC interfaced with MS with anelectrospray ion source allows the separation andaccurate molecular mass determination of the individualmembrane proteins contained in each peak on the HPLCtrace, in particular, for the identification of the majorLHCII and minor (CP24, CP26, and CP29) antennasystem, whose molecular masses range between 22 and29 kDa.(85)

Table 2 reports the comparison of the molecular massvalues calculated from the protein sequence derivedfrom the isolated genes with those determined by RP-HPLC/ESI-MS together with the apparent molecularmasses given by SDS/PAGE. The molecular mass valuesdetermined are in good agreement with the computedmolecular masses of these proteins based on their DNAsequences.



Table 2 Comparison between experimental and computed values of the molecular masses(Da) of the protein constituents of the PSII major and minor antenna system

Antennaproteins

SDS/PAGE:apparent molecular

mass (Da)

Molecular mass(measured)

Molecular mass (expected)

Spinach Other speciesa

Type 1 I 27 000–28 000 24 936 25 030 25 026 (T)24 969 (M)

Type 1 II – 24 942 – 25 041 (T)24 906 (P)

Type 2 25 000–27 000 24 761 – 24 834 (T)Type 3 24 000–25 000 24 323 – 24 285 (B)

24 308 (T)CP 29 29 000–31 000 28 076 – 27 804 (B)

27 642 (T)?CP 26 26 000–29 000 27 068 – 26 607 (M)

27 642 (T)?CP 24 20 000–22 000 22 820 22 813 –

aPlant species: P, petunia; M, maize; T, tomato; B, barley.

Thus, the assignment of each peak resolved byRP-HPLC performed as above by electrophoresis,immunoblotting, and amino acid sequencing is corrobo-rated by the values of molecular masses determined by thecombined use of HPLC coupled on-line with a mass spec-trometer equipped with an electrospray ion source (ESI-MS). Furthermore, the resolution of two variants of type Iproteins is in agreement with previous findings reportingthe resolution of more than one type I protein by bothhigh-resolution polyacrylamide gel electrophoresis(81)

and HPLC(80) and is consistent with the high copynumbers of Lhcb1 genes isolated in higher plants,(75)

giving confidence in the real potential of these coupledtechniques. On the other hand, the isolation of multiplecopy numbers of the same gene from several higher plantspecies accounts for the resolution of different variants ofthe same proteins reported in the literature.(75)

In the case of glycoproteins, ESI-MS can detect whetheran oligosaccharide is O- or N-linked.(115) It can alsodifferentiate between complex, high-mannose, and hybridforms. Moreover, this technique may be used to gainlimited linkage order information using collision-induceddissociation (CID) with both a single- and a triple-quadrupole mass spectrometer. In fact, in a complex map,the region of the map that contains the glycopeptides canbe deduced by looking for characteristic patterns in the2-D plot or by the observation of oxonium ions producedby CID. The plot of m/z against retention time (contourmaps) as a facile approach to the rapid 2-D mappingof complex samples has some similarity to the popular2-D techniques currently used in biochemistry, such as acombination of IEF and SDS/PAGE, but offers a differentcombination of orthogonal separation methods. Suchmaps are readily available from the data generated byan HPLC/MS analysis and can give valuable informationabout glycosylation patterns and product consistency.

This new analytical method can be used to deducepossible carbohydrate structures by determining both themass and elution position of individual glycopeptides.

RP-HPLC/ESI-MS has greatly expanded the powerof peptide mapping to identify carbohydrate structuresthat are attached to asparagine, serine, or threonineresidues. One can use in-source CID to scan the mapfor regions with a high concentration of glycopeptides.Such information should prove invaluable in determiningthe role in the biology of the carbohydrate moiety inglycoproteins and in reducing the approval barriers forthe pharmaceutical use of glycosylated proteins producedby mammalian fermentation systems.

6.1.3 High-performance Liquid ChromatographyCoupled On-line with Light Scattering, UltravioletAbsorbance, and Refractive Index Detectors

Since protein molecules self-associate to oligomers forspecific purposes and we often have no idea whether theprotein exists in solution as a monomer, dimer, or otheroligomer, the possibility of determining the molecularweights of proteins or their complexes is an important stepin understanding proteins and their functions. In additionto HPLC coupled on-line with a mass spectrometer,techniques of using SEC with on-line light-scattering,UV absorbance, and refractive index detectors haverecently been reported.(115) Conventional SEC is a simpleand fast method for estimating the molecular mass of aprotein in its native form based on its elution position.However, there are several problems with this simpleSEC approach. One is that the elution position dependsnot only on the molecular mass of the protein butalso on its shape. Another problem is that the elutionposition will change if the protein has any tendency tointeract with the column matrix. In addition, when a



protein or a protein complex contains carbohydrates, thecarbohydrates usually have a disproportionately largeeffect on its elution position, so SEC may not be ableto determine its polypeptide molecular mass. However,as reported by Wen et al.,(116) by using two or moredetectors, proteins and glycoproteins may be easilydetermined.

6.1.4 Affinity Chromatography and Biosensors

Affinity chromatography, which in the past yieldedsuccess as a preparative tool, has stimulated thedevelopment of analytical applications for biomolecularrecognition and most recently of molecular recognitionbiosensors. Affinity-based methods on other solid-phasesurfaces such as blots and microliter plates have furtherexpanded affinity-based technology. The principles ofanalytical affinity chromatography have recently beenadapted for a nonchromatographic affinity technology,namely, real-time optical biosensors for interactionanalysis. As a real-time method, biosensors offer theopportunity to measure not only the equilibrium affinityconstant but also the on and off rate constants forinteractions of biological macromolecules.(117)

6.1.5 High-performance Liquid Chromatography/MassSpectrometry for protein quantitation

The final step of the classic experimental proteomicsworkflow is characterized by protein/peptide identifi-cation (and/or sequencing) throughMS. For in-depthdescription of MS techniques, the interested reader isreferred to other chapters of this encyclopedia. In thiscontext, it will suffice to say that a mass spectrometerroughly includes an ionization source, a mass analyzer,and a detector. The ionization source produces gaseousions from molecules in either a solution or a solidphase. Two of the most common sources are electro-spray ionization (ESI) and MALDI (matrix-assisted laserdesorption/ionization). The mass analyzer measures themass-to-charge ratio of these ionized molecules. The mostcommon mass analyzer (time-of-flight, TOF) determinesthe mass-to-charge ratio by measuring the time requiredfor the ions to pass through a charged field.

While application of MS to identification of proteins/peptides is long known, recent trends in proteomicstarget the quantitative rather than (or in addition to)the qualitative issue. Yet it still is an almost unexploredterritory, which is of growing pivotal interest especially inthe quest for new biomarkers of pathological conditions.A detailed review about the emerging role of quantitativetechniques has been recently published.(118)

Recent applications of MS techniques allow quanti-tation of protein species in the sample under analysis,

such as cysteine labeling by isotope-coded affinity tagging(ICAT) and isotope tagging for relative and abso-lute quantitation (iTRAQ). ICAT relies on chemicallabeling reagents (ICAT reagents) consisting of threegeneral elements: a reactive group that labels a definedamino acid side chain (e.g. iodoacetamide to modifycysteine residues), an isotopically coded linker, and atag (e.g. biotin) for the affinity isolation of labeledproteins/peptides. For the quantitative comparison oftwo proteomes, one sample is labeled with the isotopi-cally light probe and the other with the isotopicallyheavy version. To minimize error, both samples are thencombined, digested with a protease (i.e. trypsin), andsubjected to avidin affinity chromatography to isolatepeptides labeled with ICAT reagents. These peptides arethen analyzed by liquid chromatography/MS. The ratiosof signal intensities of differentially mass-tagged peptidepairs are quantified to determine the relative levels ofproteins in the two samples. iTRAQ is based on the cova-lent labeling of the N-terminus and side chain aminesof peptides from protein digestions with tags of varyingmass. The samples are then pooled and usually fraction-ated by nano-liquid-chromatography and analyzed bytandem MS. A database search is then performed usingthe fragmentation data to identify the labeled peptidesand hence the corresponding proteins. The fragmenta-tion of the attached tag generates a low-molecular-massreporter ion that can be used to relatively quantify thepeptides and the proteins from which they originated.

It is relevant to underline that both the ICAT andiTRAQ approaches rely on intermediate HPLC fraction-ation methods, involving ion-exchange chromatographyand MudPIT-like strategies (2-D-RP-SCX-HPLC). Thisis relevant in the light of the renewed interest growingaround chromatographic approaches, as long as new andpivotal analytic strategies are introduced.

Although quantitative results could also be obtainedwith gel-based approaches, as, for example, changesin quantities of platelet proteins during storage withfluorophore-labeled 2-D-DIGE (differential in-gel elec-trophoresis), the obtained results are not as much asaccurate. Indeed, one approach appears to be more suitedto investigate proteins, while the other is more sensitiveto changes in peptide concentrations, making it necessaryto adopt an integrated strategy (or to choose the eligibleone) depending on the fraction of interest.

6.1.6 Posttranslational Modifications

Other than quantitative data, modern researchers aregrowingly interested in the study of posttranslationalmodifications in order to gain functional and physiolog-ical insights through protein-targeting studies. Indeed, asmany as 300 posttranslational modifications of proteins



are known to occur physiologically. Eukaryotic plasmamembrane proteins are often heavily posttranslation-ally modified in the extracellular domains (e.g. N-glycosylation) and in the intracellular domains (e.g.phosphorylation or acylation).

MS is a central technology in the protein chemist’stoolkit, enabling site mapping and quantification ofchemical modifications on proteins, as well as detection ofnew types of structures. Key to analyzing posttranslationalmodifications by MS is to understand their chemicalbehavior in solution and gas-phase reactivities, giventhat the range in chemical behavior of amino acids andfunctional groups causes significant differences amongpeptides with variable composition.

An interesting and updated review of recent strate-gies toward posttranslational modifications, in particular,glycosylations (see Sections 2.2.3 and 6.1.2), phospho-rylations, acetylations, and ubiquitinations, has beenproposed by Witze et al.(119) Although it seems to bedistantly related to chromatography, albeit tied to MS,the study of posttranslational modifications often relieson the preliminary enrichment of species of interest, suchas of phosphorylated peptides or proteins. Low sensitivityis a frequent obstacle when analyzing phosphopeptides orphosphoproteins by MS. Substoichiometric phosphoryla-tion often occurs, reducing phosphoanalyte abundancescompared to corresponding unphosphorylated forms. Inaddition, phosphopeptides may show inefficient ioniza-tion or may be lost preferentially during handling byadsorption to metal or plastics. Thus, a large repertoireof techniques has been developed to enrich phosphoan-alytes and improve detection sensitivity, particularly forsamples of high complexity. Many of these make useof reactive chemistries for covalent coupling or affinitypurification. The latter approach includes immobilizedmetal-affinity chromatography (IMAC), ion-exchange-HPLC, and antibody-based affinity chromatography.The IMAC method has been shown to facilitate therecovery, sequencing, and assignment of hundreds ofphosphopeptides from complex protein samples, such asplant membrane proteins (for review, see the article byJensen(120)).

6.2 Lipoproteins

CCC is the generic name for various liquid–liquidpartition chromatographic methods used without solidsupport matrices.(121) The stationary phase is retained inthe column with the aid of gravity or centrifugal force. Inthis way, the system eliminates all complications arisingfrom the solid support. Previous studies have shownthat a mixture of HDL and LDL fractions preparedby ultracentrifugation could be separated by x-axisultracentrifugation.(122) Recently, the complementary use

of CCC was attempted for the separation of three mainclasses of lipoproteins. Consequentely, the fractionationof HDLs, VLDLs, and LDLs may be performed by thecombined use of polymer-phase CCC and hydroxyapatitechromatography without prior ultracentrifugation.(123)

In the last decade, CE has emerged as a powerfulseparation technique for separation of plasmaapolipoproteins.(124) By adding the surfactant SDS tothe separation buffer, the main apolipoproteins of HDL(apoA-I and apoA-II) and LDL (apoB) may be separatedin a single run in <12 min. The CE separation of VLDLapolipoproteins has also been studied. High-performancecapillary isotachophoresis (ITP) of lipoproteins has beenreported, which provided for rapid screening of lipopro-tein abnormalities.(125)

7 COMPARISON WITH OTHER METHODS

To summarize, it is not surprising that HPLC techniques,given the wide versatility, relative ease of use, and highresolution, may be considered the most valuable tool forthe characterization of virtually any hydrophobic protein.The great advantage of the HPLC method lies in the factthat it can be hyphenated on-line to ESI-MS to obtainintact mass data suitable for protein identification. Theseparation of each protein by HPLC is far superior tothat possible with SDS-PAGE, and the identification ofproteins is easy and quick by intact mass measurements,avoiding trypsin digestion and mass fingerprinting ofproteins, which is often not sufficient for unequivocalprotein identification because of the limited number ofpeptides obtained from these hydrophobic proteins. Ourobservations that the chromatographic behavior of thePSI antenna proteins in RP-HPLC is similar in all speciesinvestigated along with the strong correlation revealedbetween experimental retention times of the antennaproteins examined and their predicted values based onthe overall hydrophobicity and polypeptide chain lengthmean that RP-HPLC could probably be used to provideaccurate and reliable fingerprints of proteins preparedfrom different species, without resorting to proteinidentification by additional methods. A further benefitderives from the fact that the peak area of chromatogramsis proportional to the quantity of the separated sample,so the method may be suitable for estimating thestoichiometry of proteins. This provides an attractivealternative technique with which to monitor the subtlechanges that often accompany physiological adaptationsin terms of concentration of components and addition ofchemical modifications, such as phosphorylation, throughthe increase in intact molecular mass. In the particularcase of thylakoid membrane, the ability to simultaneouslyidentify and quantify a large number of proteins, such as



most of the components of PSI and PSII, will greatlyfacilitate investigation of stress-induced changes in thestoichiometry of the supramolecular complexes.

New supports offer a good compromise betweenmechanical stability and nonspecific binding, as wellas an excellent recovery of the protein loaded. More-over, these matrices also have excellent chemical stabilitywithin the working range of biological separations. Inthis context, the use of a RP C-4 column, where theinteraction of proteins with the matrix is very soft, hasallowed a good separation and the complete recoveryof proteins when applied to the separation of photosyn-thetic apparatus.(77) In this particular case, where theprotein molecular masses are similar and the aminoacid sequence is sufficiently conserved, electrophoresisand immunoblotting appeared expensive and techni-cally demanding. Moreover, for quantitative analysis,traditional approaches by SDS/PAGE are not onlycumbersome but also rather ineffective for evaluatingdifferences in the relative quantity of each component,unless time-consuming antibody titration is used.(126)

Recent availability of high-resolution scanners and highlysensitive colorations and labeling approach (for example,through fluorophore-linked CyDyes, which is at the basisof the success of high-performance differential gel elec-trophoresis using fluorescent dyes (DIGE)) have partiallyhelped overcoming this issue hampering broader diffusionof gel-based approaches.(62) However, recent introductionof quantitative approaches involving HPLC-MS makesit desirable to pursue quantitation through the chro-matographic/mass spectrometric workflow streamline,especially in the fields of proteomics and metabolomics.

In Table 3 are summarized the advantages anddisadvantages of separations performed by HPLC withother alternative separation systems such as SDS/PAGE,IEF, and CE. The number of reports dealing with theseparation of membrane proteins by high-performanceCE is still rather small. First results indicate thatvirtually all methods used for the separation of water-soluble proteins can also be applied to the separationof hydrophobic membrane proteins. Apart from theguidelines mentioned above, the know-how acquired

in high-performance CE and in the preparative free-flow (ITP) of serum lipoproteins can be transferredto the separation of membrane proteins.(127) We havedemonstrated that capillary zone electrophoresis (CZE)can be successfully applied for the complete resolutionof the LHCII thylakoid membrane proteins by usingthe neutral detergent OD at a concentration lowerthan its critical micelle concentration in the electrolytesolution.(128) This method was revealed to be rapid andsensitive for identifying and determining quantitativelythe several components of PSII, but the RP-HPLCmethod,(77) in addition to being rapid, simple, and precise,has proven to be effective at detecting differences in theprotein components of LHCII isolated from differentplants that might not be evidenced by denaturingSDS/PAGE, as in the case of many species (data notshown). In addition, the possibility of separating allprotein components of the PSII major and minor antennasystems in samples not subjected to sucrose gradientultracentrifugation, as in the case of injection of BBY, isexpected to be advantageous for evaluating the relativecontent of the different protein components of PSIIand their variation related to physiological adaptationto environmental conditions. The injection of BBYdirectly allows one to obtain an exact evaluation ofthe quantitative relationships between chlorophyll a/b

binding present in PSII, avoiding fractionated separation,Coomassie brilliant blue staining, quantification bydensitometry, and correction of the results accordingto the specific binding of Coomassie brilliant blue toisolated proteins, as required in SDS/PAGE. Using thismethod in screening photosynthetic mutants and plantsadapted to different environmental conditions will helpin the elucidation of the composition and supramolecularorganization of LHCII and will possibly increase theunderstanding of the molecular mechanisms underlyingthe physiological adaptations.

As shown in the application examples in Section 5,all HPLC methods developed for membrane proteinseparations offer the further advantage of analyzingthe content of each HPLC peak by a multidimensionalapproach. A single peak observed in the RP-HPLC

Table 3 Comparison of the advantages and disadvantages of separations performed by HPLC and other methods

Parameter RP-HPLC CZE IEF SDS/PAGE BN-PAGE

Analysis time 30 min 10 min Hours 18–24 h 18–24 hReproducibility Excellent Reasonable Operator Operator Operator

dependent dependent dependentSample amount (µL) 1–50 1–5 1–100 1–100 1–100Resolution Good Good Excellent Excellent ExcellentSensitivity Nanograms Picograms Micrograms Micrograms MicrogramsQuantitative Excellent Good Reasonable Reasonable Reasonable

analysisAutomation Yes Yes No No No



separation of a protein is not an indication that theprotein is highly pure because it is common for relatedsubstances to co-elute with the parent protein. Themain peak can be isolated and reinjected into analternative separation system such as an SDS/PAGE,IEF, CE, or dissimilar HPLC system, taking appropriatemeasures to avoid decomposition of the protein aftercollection from the HPLC system. If the HPLC solventsystem is not compatible with the second separationsystem, or if suitable stability of the isolated mainpeak is not attainable, then the nonfractionated proteinproduct can be subjected to one of the alternativeseparation procedures. On-line spectroscopic monitoringusing diode-array detection can be of value in establishingpeak purity in favorable cases, but usually protein spectraare too similar to provide much discrimination.

The RP-HPLC/ESI-MS method holds several advan-tages over SDS/PAGE, the conventional technique forstudying membrane proteins, including better proteinseparation, mass accuracy, speed, and efficiency. Ourstudy has shown that RP-HPLC/ESI-MS is an effectivemethod for separating and characterizing the integralmembrane proteins comprising the PSII major andminor antenna systems, both as isolated complexes bysucrose gradient ultracentrifugation and as the BBYgrana membrane preparation directly. In accordancewith the molecular genetic data reported in the liter-ature, showing that higher plants have several Lhcb1genes encoding different type I proteins for each species,two type I proteins of similar molecular mass havebeen resolved in spinach leaves. The experimental dataare in good agreement with the molecular masses ofthe individual antenna proteins calculated on the basisof their nucleotide-derived amino acid sequences. Inaddition, the RP-HPLC/ESI-MS method allows the sepa-ration of protein constituents of the major and minorantenna systems, which are not resolved by conven-tional SDS/PAGE methods. Other advantages of RP-HPLC/ESI-MS over SDS/PAGE include the accuracy indetermining the molecular mass and the higher speed andefficiency. However, recent advancements in the field ofgel-based approaches have made it possible to rely onalternative methods for separation of more hydrophobicproteins, mainly in their native conditions and as aggre-gates (for a recent review, the interested reader is referredto Helbig et al.(3)). Nonetheless, these techniques arestill rather underdiffused and require a specific technicalexpertise.

The use of chromatographic methods offers the possi-bility of combining more techniques, and therefore, bothSEC and RP-HPLC methods will be employed to providehigh-resolution, quantitative data, in addition to variousspectroscopic, electrophoretic, and immunochemicalprocedures for more specialized purposes. Conventional

electrophoretic techniques often do not directly add to theinformation obtained because HPLC frequently offersbetter resolution of related substances. In this respect,2-D-HPLC approaches involving multiple dimensionseparation of protein species through chromatographyhave further expanded the role of chromatographic tech-niques in the protein-oritented area of investigations,giving birth to the so-called shotgun proteomics approach.Indeed, recently introduced MudPIT approaches havecontributed to make HPLC one of the eligible strategiesfor protein separation, and also for the more hydrophobicfraction.(11)

However, a key advantage of conventional elec-trophoretic techniques is the ability to detect virtuallyany protein, thereby ensuring that any unanticipatedprotein impurities would be detected. In developing theHPLC methods to be used in the battery of tests, onemust also consider the information obtained from theother techniques and seek to understand the relation-ship between these sets of information. The objective ofsuch an assessment is to obtain the maximum amount ofrelevant information consistent with an efficient use ofanalytical resources.

Concerning lipoproteins separation, it was reported inSection 6 that by using CE, high separation efficienciesand short analysis times for lipoproteins can be achieved.CE exhibits advantages over HPLC for lipoproteinanalysis: CE separations are faster and do not requirethe use of expensive columns, and CE does not sufferfrom slow mass transfer rates, which lead to bandbroadening in HPLC separations of apolipoproteins. Themain advantage of CE over conventional electrophoresisis speed and instrumental format, which eliminates theneed for labor-intensive steps such as gel preparation andstaining.

Structural studies on proteins depend on an inves-tigator’s ability to isolate and purify the protein. Inmany cases, protein isolation is a trivial matter, butpurification to homogeneity is a lengthy process. Tradi-tionally, proteins have been purified by a combinationof precipitation; open-column chromatography, includingSEC and ion-exchange chromatography; ultracentrifuga-tion; and electrophoresis. HPLC has become increasinglyimportant in the purification and analysis of proteins.(129)

Brilliant strategies targeting membrane protein structuresthrough X-ray crystallography have helped to accumu-late knowledge on membrane proteins. Indeed, structuralinformation is as much as fundamental as qualitativeand quantitative results that can be obtained throughHPLC (and MS) analyses. These information are rele-vant not only to understand conformational variationsbut also to build up reliable models, which reflectthe functional role of a specific membrane protein orcomplex, as in the case of plant light-harvesting complex



Figure 14 Stereo diagram of the temperature factor distribution in the structure of spinach LHCII (PDB code 1RWT). Thecolor gradient ranges from blue (lower-temperature factors, rigid) to red (higher-temperature factors, flexible). The only part ofthe structure with a high-temperature factor, indicating significant flexibility, is the protruding part of Neo (arrows). The asteriskindicates Chl 2. Detergent, lipids, and phytyl chains have been omitted for clarity. The approximate position of the lipid bilayer isindicated in gray. (Reproduced from Ref. 130. Nature Publishing Group, 2009.)

in spinach (Figure 14).(130) In these very cases, HPLCcould be extremely helpful in preliminary purification(and purity assessment steps), which are fundamental toguarantee optimal crystallization and subsequent struc-tural analyses.(130)

Finally, recent trends in protein-oriented studiesaim at determining quantitative and posttranslationalmodification changes in biological samples. In thisrespect, chromatography appears to be the most suitableanalytical method to be performed before final MSanalysis.

ACKNOWLEDGMENTS

I am grateful to former and present members of theresearch group who have contributed to much of the workpresented in this review and to Dr Anna Maria Timperioand Dr Sara Rinalducci for their valuable assistance in thepreparation of the manuscript. This work was supportedby CE Project CIPA CT93 0202 and COST Contract ERBIC15CT 980126.

ABBREVIATIONS AND ACRONYMS

AIDS Acquired Immune DeficiencySyndrome

BLM Perfusion Planar LipidMembrane

BN Blue NativeBN-PAGE Blue Native Polyacrylamide

Gel ElectrophoresisCCC Countercurrent

Chromatography

CE Capillary ElectrophoresisCHAPS 3-[(3-Cholamidopropyl)

Dimethylammonio]-l-propanesulfonate

CHD Coronary Heart DiseaseCID Collision-Induced

DissociationCN ) Clear NativeCN-PAGE Clear Native Polyacrylamide

Gel ElectrophoresisCPG Controlled Pore GlassCTAB-PAGE Cetyltrimethylammonium

Bromide Polyacrylamide GelElectrophoresis

CZE Capillary ZoneElectrophoresis

DDM n-Dodecyl β-D-maltoside2-DE Two-Dimensional

Electophoresis2-D-HPLC Two-Dimensional

High-Performance LiquidChromatography

2-D-IEF/SDS-PAGE Two-DimensionalIsoelectrofocusing/SodiumDodecyl SulfatePolyacrylamide GelElectrophoresis

DIGE Differential in-GelElectrophoresis

DM Dodecil Maltosideβ-DM n-Dodecyl-β-D-maltosideDTT DithiothreitolEDTA Ethylenediaminetetraacetic

AcidEGTA Ethylene Glycol

Bis(β-Aminoethyl



Ether)-N ,N ,N ′,N ′-TetraaceticAcid

ELISA Enzyme-LinkedImmunosorbent Assay

ESI Electrospray IonizationESI-MS Electrospray Ionization Mass

SpectrometryFA Formic AcidFFE Free Flow ElectrophoresisFPLC ) Fast Protein Liquid

ChromatographyGE Gel ElectrophoresisGPC Gel Permeation

ChromatographyHDL High-Density LipoproteinHEPES N -(2-

Hydroxyethyl)Piperazine-N -(2-EthanesulfonicAcid)

HIV Human ImmunodeficiencyVirus

HLP HyperlipoproteinemicHPLC High-Performance Liquid

ChromatographyHPLC/MS High-Performance Liquid

Chromatography/massSpectrometry

IA ImmunoaffinityICAT Isotope-Coded Affinity

TaggingID Internal DiameterIEF Isoelectric FocusingIMAC Immobilized Metal-Affinity

ChromatographyITP IsotachophoresisiTRAQ Isotope Tagging for Relative

and Absolute QuantitationLC Liquid ChromatographyLDL Low-Density LipoproteinLHCI Light-Harvesting Complex of

Photosystem ILHCII Light-Harvesting Complex of

Photosystem IIMAb Monoclonal AntibodyMALDI Matrix-Assisted Laser

Desorption/ionizationMES Morpholinoethanesulfonic

AcidMS Mass SpectrometryMudPIT Multidimensional Protein

Identification TechnologyNADP Nicotinamide Adenine

Dinucleotide PhosphateOD n-Octyl β-D-glucopyranoside

PMSF PhenylmethanesulfonylFluoride

PS-DVB Polystyrene/divinylbenzenePSI Photosystem IPSII Photosystem IIRP Reversed-PhaseRP-HPLC Reversed-Phase


RT Room TemperaturesCD4 Soluble Cd4SCX Strong Cation-ExchangeSCX-HPLC Strong Cation-Exchange


SDS/PAGE Sodium Dodecyl SulfatePolyacrylamide GelElectrophoresis

SDS Sodium Dodecyl SulfateSE-HPLC Size-Exclusion


SEC Size-ExclusionChromatography

TFA ) Trifluoroacetic AcidTIC Total Ion CurrentTOF Time-of-FlightUV UltravioletVLDL Very Low-Density

Lipoprotein

RELATED ARTICLES

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Peptides and Proteins (Volume 7)Capillary Electrophoresis in Peptide and Protein Anal-ysis, Detection Modes for • Capillary Electrophoresisof Proteins and Glycoproteins • Capillary Elec-trophoresis/Mass Spectrometry in Peptide and ProteinAnalysis • Gel Electrophoresis in Protein and PeptideAnalysis • Miniaturization of High Performance LiquidChromatography Separations and Equipment in Peptideand Protein Analysis • High-performance Liquid Chro-matography/Mass Spectrometry in Peptide and ProteinAnalysis • Reversed-phase High-performance LiquidChromatography in Peptide and Protein Analysis

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