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Chromatography of Membrane Proteins and LipoproteinsLello Zolla and Angelo DAlessandro University of Tuscia, Viterbo, Italy1 Introduction 2 Chromatography of Membrane Proteins 2.1 Methods for Protein Separation and Characterization 2.2 Separation Depending on the Membrane Protein Category 3 Chromatography of Lipoproteins 3.1 Methods of Lipoprotein Separation 4 Electrophoresis of Membrane Proteins: A Brief Overview 4.1 Classical Gel-based Approaches 4.2 Native Gel-based Approaches 5 Examples of Application 5.1 Experimental Considerations 5.2 Membrane Proteins 5.3 Separation of Lipoproteins 6 Current Trends 6.1 Membrane Proteins 6.2 Lipoproteins 7 Comparison with Other Methods Acknowledgments Abbreviations and Acronyms Related Articles References 1 2 3

tool for the characterization of virtually any hydrophobic protein. Application examples are described, and comparisons with other methods are discussed. Moreover, HPLC is not a destructive technique, and therefore, proteins, once separated, are available for further analytical investigations. Among these techniques, quantitative and qualitative analyses of the separated fractions can be obtained through other biophysical approaches, such as crystallography or structural spectroscopy. Most of these approaches require preliminary protein purication (90% or higher), which could be rapidly obtained through preliminary HPLC.

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INTRODUCTION

The available methods for the separation of membrane proteins and lipoproteins are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, isoelectric focusing (IEF), and capillary electrophoresis (CE), along with the recently introduced gel-based native techniques (blue native (BN) and clear native (CN)), and high-performance liquid chromatography (HPLC). In this article, it is shown that HPLC techniques, 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 of Analytical Chemistry, 2000, John Wiley & Sons Ltd. Tel.: +39 0761 357 100; fax: +39 0761 357 630. E-mail address: zolla@unitus.it

The importance of membrane proteins is highlighted by the fact that about one-third of all the genes in various organisms code for this class of proteins.(1) Over two-thirds of all medications exert their effects through membrane proteins, which makes them a major target of pharmacological interest.(2) However, approaches targeting membrane proteins are hampered by unminor technical challenges. Owing to their lipophilic character, the solubilization and separation of membrane proteins and lipoproteins normally requires the use of detergents. Consequently, classical protein purication strategies, designed for water-soluble proteins, are of limited value, and the number of reports dealing with the separation and characterization of such proteins is limited. Moreover, their hydrophobic nature induces selfassociation into noncovalent multimers, and therefore, all separative methods require preliminary procedures such as tedious sequential gradient ultracentrifugation for sample preparation, with the risk of affecting the results. Although the available methods for the nal separation of these hydrophobic proteins are numerous, their high hydrophobicity and the presence of detergents make most of these available methods expensive and technically demanding.(3) The traditional approaches by SDS/PAGE are not only cumbersome but also rather ineffective for evaluating differences in small molecular masses, and the run times required are very long, typically more than 2030 h. More complex gelbased approaches, such as two-dimensional isoelectrofocusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D-IEF/SDS-PAGE) hold a great separation potential. Although two-dimensional electophoresis (2-DE) has many benets, the technique does not lend itself to large-scale, high-throughput proteomic analyses, as not all types of proteins are well resolved in this system. Proteins bearing extremes of size, hydrophobicity, or charge fail to enter the gel and are not or underrepresented.(4) Although membrane proteins

Encyclopedia of Analytical Chemistry, Online 20062011 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

2with up to 12 transmembrane -helices have been resolved and identied by 2-DE-MS (mass spectrometry), most membrane proteins have been resistant to this approach.(3) CE could represent a powerful separation technique for lipoproteins, but the number of reports on membrane proteins is still limited. It is not surprising that HPLC techniques, given their wide versatility, relative ease of use, and high resolution, may be considered the most valuable tool for the characterization of virtually any hydrophobic protein.(5) Moreover, HPLC is not a destructive technique, and therefore, proteins, once separated, are available for other analytical investigations, such as by SDS/PAGE, or other biophysical analyses, such as crystallography or structural spectroscopy. Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet 50 000, whereas the amount of polypeptide of Mr < 15 000 that can be loaded onto the porous stationary phase is approximately 10-fold that of the monolithic one.(90) The reason for this discrepancy is the difference in accessibility of the chromatographic surface to analytes of different molecular size. The surface in the micropores of porous separation media is not accessible to large biomolecules; thus, the loading capacity decreases rapidly with increasing molecular mass. Moreover, compared to octyl or octadecyl stationary phases, PSDVB monolithic stationary phases are known to be mildly

PEPTIDES AND PROTEINS

hydrophobic(91,92) , which makes them eminently suitable for the separation of very hydrophobic membrane proteins. In this review,(93) the applicability of the RPHPLC/ESI-MS method (using both monolithic capillary and beaded columns) to the analysis of larger proteins in biological samples in a difcult matrix is demonstrated using the protein components of PSI of thylakoid membranes whose molecular masses range between 3500 and 80 000. Figure 9 compares the separation of all PSI components performed by capillary monolithic and a beaded silica capillary lled with C4 RP. The separation time of the monolithic column is almost halved compared to that expected of the same protein mixture on a conventional, butyl-silica stationary phase with 300-A pores, and the resolution of the peaks is signicantly better. The peak widths at half-height ranged from 3 to 5 s, which demonstrates the high resolving power and speed of analysis with monolithic PS/DVB capillary columns. However, in both cases, mass spectra of high quality were extracted from the reconstructed ion chromatograms, showing no adduction with detergent. Interestingly, the elution order changed from Lhca1< Lhca3< Lhca4< Lhca2< PsaF on the silica-based stationary phase to Lhca1< Lhca4< Lhca3< PsaF< Lhca2 on the monolithic column, which points to differences in selectivity between the silica-bonded and the polymeric stationary phase. 5.2.8 Reversed-phase High-performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry of Entire Thylakoid Membranes In this last section, we present the results of a study designed to develop a rapid and straightforward method, using HPLC, to resolve and identify the protein components of both the PSI and PSII, starting from the entire thylakoid membrane. Normally, before loading a complex biological matrix on any HPLC system, membrane proteins must be preseparated according to their hydrophobic characteristics, and this is achieved by selective extractions. This pretreatment facilitates subsequent separation and provides a rst guideline for the choice of detergent. As a rule, the detergent chosen for the running buffers is the same one with which the protein was solubilized. Also, in order to retain the biological activity of proteins, it is preferable to use less denaturing detergents and nonionic detergents for solubilization and in the running buffers (the interested reader is referred to the article by Seddon et al.(8) for further details on the use of detergents for membrane protein analysis). The presence of detergent in all steps minimizes the tendency toward association and aggregation as well as the possibility of nonspecifc interactions with the support used for chromatographic separation.

Encyclopedia of Analytical Chemistry, Online 20062011 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

CHROMATOGRAPHY OF MEMBRANE PROTEINS AND LIPOPROTEINSLhca4 Lhca2.1 Lhca2.2

21PsaA: PsaB PsaL

Absorbance at 214 nm

120PsaC; PsaE; PsaN Lhca3 PsaG

PsaH

PsaD

Lhca1.1 Lhca1.2

80

PsaK

40

0 0 1.2psa N

(a)

10

20

30

40

PsaF

50

60

Absorbance at 214 nm

psa D

lhca 3 psa F lhca 2.1 lhca 2.2

psa H psa C psa E

lhca 1.1 lhca 1.2

psa G

lhca 4

0 (b) 0 10 20 Time (min) 30 40

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% triuoroace

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