biotinylation reagents for the study of cell surface proteins

REVIEW

Biotinylation reagents for the study of cell surface

proteins

Giuliano Elia

Mass Spectrometry Resource, Conway Institute of Biomolecular and Biomedical Research,University College Dublin, Belfield, Dublin, Republic of Ireland

The extraordinarily stable, non-covalent interaction between avidin and biotin is one of the mostcommonly exploited tools in chemistry and biology. Methods for derivatization with biotin of avariety of molecules (in particular, proteins) have been introduced, in order to allow their efficientrecovery, immobilization and detection with avidin-based reagents. The field has evolved veryrapidly and the applications have become more and more sophisticated. Cell surface proteinstudies have enormously benefited from refinements of this technology. It is now possible tospecifically biotinylate one single membrane protein or to fish out a membrane receptor boundto its ligand. The release of biotinylated molecules from the avidin-based reagents, however, maystill represent a major problem, due to the stability of the complex. This review will examine thebiotin–avidin technology for the study of cell surface proteins, discussing reagents and tech-niques as well as examples of applications in quantitative proteomics.

Received: February 1, 2008Revised: April 1, 2008

Accepted: April 16, 2008

Keywords:

Avidin / Biotin / Biotinylation / Cell Surface Proteins

4012 Proteomics 2008, 8, 4012–4024

1 Introduction

In 1927, Margaret A. Boas, a research fellow at the Depart-ment of Experimental Pathology of the Lister Institute inLondon, observed that rats fed large quantities of raw eggwhite developed severe dermatitis, indicating malnutritionwhich eventually caused death [1]. Vitamin H, whose prop-erties and structure were later found identical to those ofbiotin [2, 3], prevented such dermatitis. The malnutritionwas eventually attributed to the depletion of biotin by anunknown factor, proteinaceous in nature, to which the nameof avidalbumin was initially given [3]. This protein present inconsistent amount in the egg white was able to form a verystable complex with the vitamin and interfered with the ani-mal’s nutrition.

Already in 1941, the extraordinary affinity of the protein,renamed avidin, for biotin was recognized [4]. In 1942, crys-tallization of avidin in a pure form was accomplished [5] and itbecame rapidly clear that the interaction of the relatively smallvitamin H molecule with the egg-white glycoprotein avidinand with the related bacterial protein streptavidin (SA) [6]could be easily converted in a affinity-based tool for severaldifferent purposes. Since then, and for the following decades,chemical modification of a variety of molecules with biotinhas been exploited as one of the most useful tools in bio-chemical and biomedical research. Biotinylated molecules(e.g., proteins, DNA, RNA, etc.) can easily be detected with SAderivatives (e.g., fluorophore-, horseradish peroxidase-, oralkaline phosphatase-conjugates), or efficiently captured onavidin/SA-coated solid supports (e.g., resins, magnetic beads,microtiter plates, chips). However, due to the very high stabil-ity of the biotin–SA complex (Kd , 10215 M) [7], the elution ofbiotinylated molecules from SA-coated surfaces has repre-sented a real challenge for many years.

This article will review the biotin–avidin technology forthe study of cell surface proteins, providing examples of bothcommercially available and laboratory-developed reagents, aswell as techniques for elution of biotinylated proteins fromavidin and SA. Alternative metabolic techniques for protein

Correspondence: Dr. Giuliano Elia, Mass Spectrometry Resource,Conway Institute of Biomolecular and Biomedical Research, Uni-versity College Dublin, Belfield, Dublin 4, Republic of IrelandE-mail: [email protected]: +353-1-7166703

Abbreviations: DST, disuccinimidyl tartrate; EDC, LC, long chain;NHS, N-hydroxysuccinimide; PEO, polyethylene oxide; PFP, pen-tafluorophenyl; SA, streptavidin; TFP, tetrafluorophenyl

DOI 10.1002/pmic.200800097

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2008, 8, 4012–4024 Technology 4013

biotinylation and stability of biotinylated proteins in biologi-cal samples will also be discussed. Reference to some appli-cations in quantitative proteomics will also be given.

2 Biotinylating reagents

A chemical reagent to be used for protein biotinylationexperiments is made up of the following building blocks:

(i) The biotin moiety, for the subsequent interaction ofbiotinylated proteins with avidin/SA-based reagents; thevaleric acid side chain of the biotin molecule is important inthe interaction with avidin, but its terminal carboxylic groupcan be derivatized to incorporate various reactive groups thatare used to attach biotin to other molecules. The use of biotinin conjunction with SA yields the highest binding affinityand allows the use of strong detergents during the purifica-tion of relatively insoluble proteins.

(ii) A spacer of sufficient length to allow protein captureby immobilized SA. The spacer is possibly the most impor-tant variable, in that it largely determines the properties (e.g.,solubility) of the biotinylating reagent and of the derivatizedproteins. The spacer can also be cleavable by chemical orphysical agents to facilitate protein release after capture.

(iii) A reactive moiety for the covalent binding of biotin tothe protein(s). The most common reactive groups employedinclude reactive esters, like the N-hydroxysuccinimide (NHS)group, which undergoes a nucleophilic substitution reaction inthe presence of primary amines (e.g., e-amino group in exposedlysine residues in proteins), or reactive imides, for instance themaleimido group, which reacts with free thiol groups (like theones contained in cysteine residues of proteins).

The importance of these three different building blocksis discussed in the following section.

2.1 Biotin, biotin derivatives, and their synthesis

The empirical formula of biotin was determined by du Vig-neaud et al. in 1941 [8] and its structure was established bythe same group in 1942 [9, 10]. This structure was confirmedby the first total synthesis of racemic biotin in the Merck Re-search Laboratories [11]. Finally, in 1966, X-ray crystal-lographic analysis established the absolute configuration ofnatural (1)-biotin as 1 (Fig. 1) [12]. To date, more than 40different synthetic pathways for biotin have been proposed.They have been exhaustively reviewed in ref. [13].

As said, the valeric acid side chain is used to conjugatebiotin to other chemical groups, taking advantage of the free

Figure 1. Absolute configuration of natural (1)-biotin.

terminal carboxylic group. In initial studies in which theconcepts for biotinylating proteins were first laid down, bio-tinyl-NHS ester (NHS-biotin) was used for incorporating thebiotin moiety via lysines into several bacteriophages, lectins,and antibodies [14–17]. NHS-biotin was obtained by directcoupling of biotin and NHS in the presence of dicyclohex-ylcarbodiimide in dimethylformamide (DMF) [18]. NHS-biotin has been for long the most popular biotinylatingreagent and has been used almost exclusively for biotinyla-tion of proteins. Reasons for its widespread usage are thatlysine residues are numerous in most proteins and that theyfrequently occupy an accessible position. Furthermore, thelysine residues are usually not directly involved in proteins0

biological activity, and their modification generally has littleeffect on the interaction of a protein with its substrate. Theutility and efficiency of action of this reagent are underlinedby the dozens of commercial enterprises (Pierce, MolecularProbes, Invitrogen, Quanta BioDesign, to name but a few)that include NHS-biotin in their catalogs.

In recent years NHS-biotin has been replaced withlonger-chain homologs, e.g., biotinyl-e-aminocaproyl-NHS(NHS-long chain (LC)-biotin) ester, in order to overcomesteric hindrance problems and ameliorate the accessibility ofthe biotin residue to avidin/SA-based reagents. Preparationsof NHS-LC-biotin start from NHS-biotin in DMF, which isreacted with bicarbonate solution of e-aminocaproic acid.The crystallized biotinyl-e-aminocaproic acid is then reactedwith NHS as described above for NHS-biotin.

Water-soluble analogs (i.e., N-hydroxysulfosuccinimidederivatives of biotin; Sulfo-NHS-LC-biotin) have also sub-stituted for the conventional biotinylating reagent. In addi-tion to NHS-biotin, other active esters of biotin (e.g., biotinyl-p-nitrophenyl ester (BNP) and its homologs) are also efficientreagents for incorporating biotin into proteins via aminogroups of lysine [19].

In 1981, Orr [20] introduced the use of 2-iminobiotin,the cyclic guanidino analog of biotin, in cell surface proteinstudies making use of immobilized avidin to recover thelabeled components, uncontaminated by other cytosolicand membrane components. The pH-dependent interac-tion of 2-iminobiotin with avidin facilitates recovery. Athigh pH, the free base form of 2-iminobiotin retains thehigh affinity specific binding to avidin characteristic ofbiotin, whereas at acidic pH values, the salt form of theanalog interacts poorly with avidin.

Iminobiotin-containing biotinylating reagents arecommercially available (e.g., from Pierce and Perkin-Elmer) but are less widespread than their biotin-con-taining counterparts, also due to the reduced tolerance todetergents of the avidin–iminobiotin complex [20].

2.2 Spacer groups

At the end of the 1980s it became apparent that, for sterichindrance reasons, interactions between avidin and a bioti-nylated protein could be dramatically improved by increasing


4014 G. Elia Proteomics 2008, 8, 4012–4024

the length of the spacer arm that connects biotin to the bulkof the biomolecule.

Sulfo-NHS-LC-biotin mentioned above (spacer armlength 8.8 Å) is probably the most popular biotinylationreagent currently available on the market. To increase spacerlength, Pierce (and other companies) proposes also a Sulfo-NHS-LC-LC-biotin (spacer arm 20.4 Å), in which two ali-phatic aminocaproic acid chains are arranged head-to-tailbetween the biotin and the sulfo-NHS group. Although thesemolecules are soluble in water, the nature of the LC linker issubstantially hydrophobic. Once labeled, the LC-biotin willseek hydrophobic regions in the protein and hide in them,making it less available to avidin. At the same time, thehydrophobic LC-biotin labels may cause serious agglutina-tion and precipitation problems.

Monodisperse PEG-based spacer arms are now availablefrom different companies (e.g., Pierce, EZ-Link PEO-biotin;Quanta BioDesign, discrete PEG™; KPL, Surelink™). Mono-disperse PEG compounds and their derivatives are suitablefor a wide range of therapeutic, diagnostic, and molecularengineering applications. Spacer lengths of up to 46 Å havebeen introduced for labeling and capture of residues deeplyburied in folded protein regions and are commercially avail-able (Quanta).

PEG-based spacers are preferred substitutes to aliphaticmethylene chain spacers in that these linkers are extremelywater soluble and hydrophilic. In addition to being watersoluble, the PEG linkers are also soluble in organic sol-vents, like DMF and DMAC, but especially in methylenechloride.

Agglutination data have compared the Sulfo-NHS-LC-biotin with NHS-dPEG4-biotin (Quanta), which has thedPEG4 spacer (comparable in length to Sulfo-NHS-LC-LC-biotin). The data shows that human IgG biotinylated with thesulfo-NHS-LC biotin precipitates within a couple of weeks,while human IgG biotinylated with NHS-dPEG4-biotinshows no agglutination on the third week [21].

It should also be mentioned that PEG-based spacers havebeen shown to be less immunogenic than aliphatic chain-based ones in in vivo applications [22]. Using a carrier proteinconjugated to a peptide by a linker is a widespread approachto elicit a peptide-specific immune response. However, theuse of conventional linkers contributes to generate a linker-dependent response. Alkyl linkers containing more than twoor three methylene groups are highly immunogenic [23],sometimes requiring the alkyl spacer length to be shortenedto reduce this undesirable property. In contrast, PEG is wellknown to be nonimmunogenic and in the above sensedPEG-based linkers are nonimmunogenic as well [21].

Cleavable spacers have been introduced and madecommercially available with the aim of facilitating therelease of biotinylated proteins after capture on immobi-lized avidin. The most common cleavable group is a dis-ulfide bridge that can be broken by reducing agents likeb-mercaptoethanol or DTT. Reagents containing thiscleavable group come associated to both aliphatic chain-

based and PEG-based linkers (e.g., in Sulfo-NHS-SS-biotinor in NHS-SS-dPEG4-biotin) and have been used in anumber of studies of cell surface proteins [24–26]. Owingto the nature of the cleavable linker, care should be takenwhen using these reagents in a reducing environment.Our own experience has shown for instance that thesereagents are not compatible with in vivo biotinylationprocedures [27]. The reducing intracellular milieu is alsonot adapted to the use of disulfide bridge-containingreagents. This fact can however be turned into an advan-tage when dealing with cell surface protein studies. Infact, notwithstanding the presence of a charged groupthat should render it membrane-impermeable, sulfo-NHS-LC-biotin has been shown to be able to permeatebiological membranes, leading to sample contaminationby cytoplasmic proteins [28]. The use of a cleavable linkerin these conditions limits contamination, due to intracel-lular cleavage of the reagent [25].

We have proposed a cleavable linker based on a vicinaldiol group, which can be cleaved by mild treatment withoxidants like sodium metaperiodate [29]. It is possible tochemically synthesize this reagent with traditional organicsynthesis methods [30, 31], as depicted in Fig. 2. Alter-natively, a rapid way of producing the same biotinylatingreagent, e.g., to label lysine residues of proteins and con-taining a vicinal diol in the linker region, is to combine theuse of an homobifunctional crosslinker (a NHS diester oftartaric acid) with a biotin derivative containing a terminalprimary amine group (Fig. 3). The reaction proceeds inthree steps. First, N-(3-aminopropyl)-biotinamide tri-fluoroacetate (Fluka, Buchs, Switzerland) is dissolved inPBS pH 7.8 and disuccinimidyl tartrate (DST) (Genotech,Lausanne, Switzerland) is dissolved in the same buffer,containing 20% DMSO. The two substances are mixed in astoichiometric ratio of 1.2:1 equivalents and allowed to reactat RT for 15 min to form the intermediate 7 (Fig. 3). Thereaction mixture is then added to the protein-containingsample in PBS pH 7.8 and biotinylation allowed to takeplace at RT for 30 min. Excess unreacted 7 and DST arequenched with an excess of a primary amine-containingreagent (50 mM Tris-Cl pH 8.0).

As a proof-of-principle experiment, we biotinylated anaqueous solution of BSA and released the biotinylated pro-tein, after capture on a SA-sepharose column, by means of adilute solution of an oxidant, like sodium metaperiodate(Fig. 4). This new reagent allows investigators to biotinylateproteins in a wide range of environmental conditions,including reducing environment (e.g., the intracellularmilieu) and to release biotinylated proteins, captured on aavidin resins, simply by treatment with dilute solutions ofoxidants.

Photocleavable linkers are also commercially available.Pierce has introduced NHS-PC-LC-biotin as a unique aminereactive, photocleavable biotin analog with an extendedspacer arm. The spacer arm imparted by NHS-PC-LC-biotinis approximately 31.4 Å in length. A photocleavable 1–2



Figure 2. Organic synthesis of a NHS-LC-biotin derivative containing a vicinal diol group as cleavable linker. Tartaric acid 2 is reacted withN,N,N0,N0-tetramethyl(N-hydroxysuccinimido)uronium tetrafluoroborate 3 to yield a mixture of esters of tartaric acid, the N,N,N0,N0-tetra-methyl uronium hemitartrate 4 (IUPAC name: [(3-carboxy-2,3-dihydroxy-propionyloxy)-dimethylamino-methylene]-dimethyl-ammonium)and the succinimidyl hemitartrate 5 (IUPAC name: 2,3-Dihydroxy-succinic acid mono-(2,5-dioxo-pyrrolidin-1-yl) ester). These esters canthen be reacted with a primary amine-containing biotin derivative (N-(3-aminopropyl)-biotinamide trifluoroacetate 6) to yield the biotin-amidopropyl hemitartramide 7 (IUPAC name: 2,3-Dihydroxy-N-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-pro-pyl}-succinamic acid). With a very similar reaction, the free carboxylic group of 7 can be then in turn activated with O-benzotriazolyl-N,N,N0,N0-tetramethyluronium hexafluorophosphate (formula not shown, see [30]) and NHS or Sulfo-NHS to yield in the end the corre-sponding succinimido or sulfosuccinimido esters 8 (IUPAC names: 1-(2,3-dihydroxy-3-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoylamino]-propylcarbamoyl}-propionyloxy)-2,5-dioxo-pyrrolidine and 1-(2,3-dihydroxy-3-{3-[5-(2-oxo-hexahydro-thieno[3,4-d]imi-dazol-6-yl)-pentanoylamino]-propylcarbamoyl}-propionyloxy)-2,5-dioxo-pyrrolidine-3-sulfonate sodium salt, respectively), ready forreaction with lysyl residues in proteins.

(nitrophenyl)-ethyl moiety is attached to the biotin throughthis long spacer arm, separating the biotin from the aminereactive NHS group at the end of the molecule. The con-jugate formed with NHS-PC-LC-biotin undergoes an effi-cient photocleavage upon illumination with 300–360 nmlight [32], resulting in the rapid release of the target proteinin an unmodified form. Photocleavable biotin derivatives area useful alternative to chemically cleavable ones; yet, in someexperimental conditions, it is not easy to achieve efficientillumination of the sample.

Other types of linkers have been proposed in the litera-ture. Biocytin (N-e-biotinyl-L-lysine) is a naturally occurringbreakdown product of biotin-requiring enzymes. Synthesisof biocytin derivatives containing a propionyl group forincreasing the spacer length has been described [33]. Her-forth et al. [34] introduced an on-bead construction protocolwhich permits the simple preparation of biotin labels withcustom tailored spacers using established amide bondforming procedures. On bead synthesis leads to simpleproduct isolation because the excess of polymer-boundreagent that is applied to drive reactions to completion canbe removed by filtration. This allows for the fast and con-

venient access to different biotinylated compounds in highyield and purity.

Solid-phase synthesis of biotin-PEG derivatives is also atthe basis of a new method for the preparation of crosslinkingprobes for membrane receptors [35]. The method was suc-cessfully applied to development of a biotin-Asp-PEG-arvanilprobe for the study of cannabinoid receptors, and holds pro-mise of becoming a general method for identification ofreceptors for small molecules.

Finally, it should be mentioned that chromophoric link-ers which include a fluorescent moiety (bis-aryl hydrazone,KPL SureLink™; Cyanocobalamin, Quanta BioDesign) havealso been made available by several companies. Thesereagents provide an easy method for labeling proteins andaccurately measure total biotin incorporation with a singlestep. The number of biotins can be quantitated by spectro-photometric analysis at UV wavelengths.

2.3 Reactive groups for protein derivatization

The chemical groups that have most commonly been tar-geted for protein derivatization are the primary amino



Figure 3. Alternative generation of a vicinal diol-containing biotinylating reagent. The biotin derivative 6 (N-(3-aminopropyl)-biotinamidetrifluoroacetate), dissolved in an aqueous buffer, is reacted with the reactive diester 9 DST or disulfosuccinimidyl tartrate (DSST). Thestoichiometric ratio of the two reagents is adjusted in order to maximize the yield of the product biotinamide (sulfo)succinimidyl tartrate 8

(biotin-(S)ST) and to prevent, to the largest extent possible, crosslinking of protein molecules. The reaction product can be purified (e.g., byHPLC) for further use or the reaction mixture can be directly applied to solution of the protein(s) to be biotinylated. The biotinylation reac-tion takes place at room temperature for 30260 min and the exposed lysyl residues of the protein(s) are bound to the vicinal diol-containingbiotin derivative. Finally, the excess unreacted biotin derivatives is quenched with an excess of a primary amine-containing buffer solution(e.g., Tris-Cl 50 mM pH 8.0) and all low molecular weight contaminants are removed by gel filtration chromatography (PD-10 columns).

groups, which are abundant in the vast majority of proteinsin the form of lysine side chain e-amines and N-terminala-amines. Amine-reactive biotinylation reagents can bedivided into two groups, according to their solubility inaqueous Solutions. NHS-esters of biotin are essentially waterinsoluble. For reactions in aqueous solution, they must firstbe dissolved in DMSO and DMF, which are compatible withmost proteins at 20 % final concentration. The organic sol-vent forms an emulsion in the aqueous phase, allowing thebiotinylation reaction to proceed. These hydrophobic NHS-esters of biotin are therefore also membrane permeable.

Sulfo-NHS-esters of biotin are soluble in water up to 10mM. They are prone to hydrolysis in aqueous milieu andshould be dissolved just before their use. The water solubilityof sulfo-NHS-esters is imparted to the compound by thesulfonate group on the NHS ring, a moiety which, in turn,renders these reagents less membrane permeable. Sulfo-NHS-esters of biotin are therefore the most frequently usedas cell surface biotinylation reagents.

Alternatively, primary and secondary amino groups inproteins can be targeted by pentafluorophenyl- or tetra-fluorophenyl ester derivatives of biotin (e.g. PFP-biotin andTFP-PEO-biotin, Pierce). These molecules are more reactivetowards amino groups at a slightly basic pH and are lessprone to hydrolysis.

Another very common target for protein derivatization isthe free sulfhydryl group, found in cysteine residues. Threedifferent reactive groups can be employed to target sulfhy-dryl groups for biotinylation. The most specific method usesreactive maleimide groups, which are extremely reactive to-ward free sulfhydryls at pH 7. The reaction of maleimidewith free thiols is carried out at pH 6.5–7.5 because at higherpH values the compound cross-reacts with primary amines.High pH values also increase the hydrolysis of the mal-eimide group.

The second reactive group used to target free sulfhydrylsis the iodoacetyl moiety. Iodoacetyl-LC-Biotin is not watersoluble and must be dissolved in a solvent before use in anaqueous reaction mixture. The iodoacetyl group mainlyreacts with cysteine thiol groups at pH 7.5–8.5, resulting in astable thioether bond. At this pH, cross-reactivity withamine, thioether and imidazole groups is minimized. How-ever, and if no cysteines are available, the reaction can be di-rected at imidazoles by adjusting the pH to 6.9–7.0. Incuba-tion time must in this case be increased (up to a week). His-tidyl side chains may also take part in reactions above pH 5.0.

Finally, pyridyldithiol groups are also used to derivatizefree sulfhydryls with biotin by a mechanism of disulfideexchange, which results in the formation of a mixed disulfidebond. The reaction of HPDP-biotin ((N-[6-(biotinamido)-



Figure 4. Elution of biotinylated BSA from SA-sepharose by metaperiodate treatment or by a SA-denaturing method. (A) Biotinylated BSAprepared according to the protocol outlined in Fig. 3 was efficiently captured by SA-sepharose (compare lane 3, “flowthrough” and lane 2,“input”). No residual BSA is present in the last wash fraction (“last wash”, lane 4). A virtually complete release of biotinylated BSA occursapplying to the SA-sepharose sample the harsh conditions described in ref. [44] (lanes 5 and 6). However, BSA is also released from SA-sepharose by means of metaperiodate (lanes 7 and 8). Molecular weight markers are shown in lane 1. The same samples shown in panel Awere used to perform a Western blot experiment. Panel B shows a staining of the proteins with the reversible stain Red Ponceau. Panel Cshows the same samples after SA-horseradish peroxidase and ECL staining. The results shown in panel C clearly indicate that capture ofbiotinylated BSA by SA-sepharose is virtually complete. Indeed, the BSA amount remaining after capture (see lanes 3 in panels A and B)appears clearly not to be biotinylated (lane 3 in panel C). Furthermore, the absence of signal in lanes 7 and 8 of panel C (release by peri-odate) clearly indicates that the released BSA (see lanes 7 and 8 in panels A and B) is not biotinylated any more, as one would expect as aconsequence of the cleavage of the vicinal diol-containing linker by treatment with periodate. The biotin moiety remains in this caseassociated to the SA-sepharose.

hexyl]-3’-(2’-pyridyldithio)propionamide) with cysteine-con-taining proteins is normally performed at neutral pH but theprocess can occur in a wide range of buffers and at variouspH conditions. A useful feature of this reaction is that biotincan be released by treatment with a reducing agent, con-ferring cleavability to the biotin-protein bond.

Carboxyl groups, which can be found in protein carboxytermini, aspartate residues or glutamate residues, are alsotargets for biotin labeling, by use of amine-derivatized biotinmolecules. This reaction is mediated by carbodiimides andresults in the formation of an amide bond. The most com-mon carbodiimide cross-linker used is l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC) and thereaction is generally performed in MES buffer at pH 4.5–5.Attention must be paid not to use buffers containing primaryamines (Tris, glycine, etc.) or carboxyl groups (acetate, citrate,etc.) as they would quench the reaction. Phosphate buffersare also not recommended because they reduce the conjuga-tion efficiency, although this effect can be overcome by add-ing more EDC.

Biotin PEO-amine, biotin PEO-LC-amine and 5(biotin-amido)pentylamine are amine-derivatized biotin moleculesthat can be reacted with carboxyl groups. Furthermore, any

of the hydrazide-derivatized biotin molecules can be reactedwith carboxyls under identical conditions. Using this strate-gy may result in some polymerization of the peptide or pro-tein if the molecule has both carboxyls and primary amineson its surface. The extent of polymerization can be mini-mized by decreasing the amount of EDC and/or increasingthe amount of the biotin reagent used.

The carbohydrate portion of glycoproteins can be reactedwith hydrazide derivatives of biotin as well. Oxidation of car-bohydrate groups in glycoproteins using 10 mM periodate isused to generale reactive aldehydes from the cis-diols of avariety of sugar moieties. Aldehydes can then be reactedspecifically with a hydrazide group at pH 4–6, forming astable hydrazone linkage. At 1 mM periodate and a tempera-ture of 07C, oxidation is restricted primarily to sialic acidresidues. Sialic acid-containing carbohydrates can also bebiotinylated with hydrazide derivatives by pretreatment withneuraminidase to release the sialic acid moiety and uncover agalactose groups. The galactose is then oxidized by galactoseoxidase and selectively biotinylated with biotin-hydrazides.Temperature, pH of oxidation and the periodate concentra-tion all affect the reaction with hydrazide derivatives of bio-tin. Also, because glycosylation varies with each protein,



optimum conditions must be determined for each glycopro-tein.

The technique described above has been successfullyused to characterize cells surface glycoproteins in humanleukocytes [36].

In the unlikely hypothesis that a protein does not containany of the reactive functional groups mentioned above, itmay still be labeled using a photoreactive biotinylationreagent. These reagents are based on the presence of a chem-ically inert group (aryl azide), which becomes activated uponexposure to UV light. The half-life of the aryl nitrene inter-mediate formed is short (on the order of 1024 s) and thisreacts rapidly and nonselectively with electron dense sites,for instance by addition into double bonds. If the aryl nitreneintermediate fails to react, an internal ring expansion reac-tion occurs and the compound becomes reactive towardnucleophiles such as primary amines and sulfhydryls. Pho-toactivation and reaction with other molecules can be per-formed in a wide variety of buffer conditions. However,acidic conditions and reducing agents should be avoided be-cause they may de-activate the aryl azide group.

Table 1 summarizes some of the most widespread func-tional and reactive groups, as well as spacer properties andcharacteristics.

3 Elution of biotinylated proteins fromimmobilized avidin/SA

The very high stability of the biotin–avidin complex(Kd , 10215 M) [7] has made of the elution of biotinylatedmolecules from immobilized avidin a major challenge forthe research community.

Investigators have proposed many different approachesto overcome this problem and combine the ease of biotiny-lation reactions with suitable reversible capture/elution con-ditions, yet most of the methods available present drawbacksin certain experimental conditions. Discussing spacergroups, we have already described biotinylation reagents thatcontain cleavable linkers. Cleavable disulfide bridge-contain-ing biotinylating reagents perform well, but may showinsufficient stability in some biological fluids prior to purifi-cation. Proteins modified by means of photocleavable biotinderivatives (e.g., NHS-PC-LC-biotin, Pierce) can be releasedusing long wave UV light [32]; yet, in some experimentalconditions, it is not easy to achieve efficient illumination ofthe sample.

On the other hand, some resins on the market make useof modified (strept)avidin molecules with a lower affinity forbiotin, therefore allowing elution under milder conditions(e.g., nitrated avidin derivatives [37], which allow elution athigh pH values (CaptAvidin from Molecular Probes), ormonomeric avidin, which displays a lower binding affinityfor biotin (Immobilized Monomeric Avidin, Pierce)) [38].The use of monomeric avidin is at the basis of importantmethodologies in quantitative proteomics, such as isotope

coded affinity tag technology (ICAT) [39]. However, loweringthe affinity of (strept)avidin–biotin interaction prevents anefficient capture of biotinylated molecules in the presence ofstrong detergents, which are often essential for the solubili-zation of hydrophobic molecules (e.g., membrane proteins).

A number of alternative strategies to disrupt or reversethe biotin–(strept)avidin linkage have previously beenreported [40–42]. Many of these techniques have been devel-oped for nucleic acid technology applications, and generallyrequire harsh conditions, e.g., boiling in high salt conditionsor use of formamide and EDTA heated to 947C for severalminutes [43], to achieve partial or complete bond disruption.Not only are such conditions generally harmful to any boundmoiety (e.g., a protein or nucleic acid molecule), but thesealso result in denaturing of the SA molecules, which cannotbe reused. Moreover, since proteins can only be recoveredunder denaturing conditions these are inappropriate for thepurification of delicate proteins. However, for merely ana-lytical purposes, the use of such methods has an intrinsicvalue.

We presented a protocol [44] for the quantitative elutionof biotinylated proteins from SA-sepharose, featuring harshelution conditions and competition with free biotin. Themethod is compatible with protein analysis by SDS-PAGEand subsequent mass spectrometric analysis for proteinidentification. Our technique can easily be applied to thebiotinylation of protein extracts, intact cells, and even wholetissues and organs. The usefulness of the method is exem-plified by the recovery of biotinylated proteins from organhomogenates, obtained from mice perfused with a reactiveester derivative of biotin [27].

The group of Mathias Uhlén reported in 2005 that a shortincubation in nonionic aqueous solutions at temperaturesabove 707C can efficiently break the interaction biotin–SA[45] without denaturing the SA tetramer. Both biotin and theSA remain active after dissociation and both molecules cantherefore be reused. The efficiency of the regenerationallowed solid supports with SA to be used many times,exemplified with the multiple reuse of SA beads used forsample preparation prior to automated DNA sequencing.

Discussing this surprising finding, the authors point outthat a recent study about the 3-D structure of SA mutants hasshown that the active site is accessible by water moleculesthrough a channel of the SA molecule reaching the biotin inthe binding pocket [46]. The study suggests that the bond-breaking events are accompanied by the entry of a singlewater molecule into the binding pocket, where it serves as abridge between the SA Asp-128 carboxylate and the biotin[46].

Dissociation of biotin from the SA binding pocket inaqueous solutions at elevated temperatures is rendered lessefficient by the presence of salts. The fact that the dissocia-tion is efficiently hindered with both monovalent and diva-lent salts suggests that the interaction is stabilized with ionicmolecules and that depletion of salts allows the complex tobe dissociated [45].



Table 1. Common biotinylating reagentsa) and their properties

Biotinylatingreagent

Functional groupin protein

Reactivemoiety inbiotinylatingreagent

Bondformed

Solublein water

Linker Cleavable Membranepermeable

Comments

NHS-biotin Primary amineb) NHS Amide No None No YesNHS-LC-biotin Primary amine NHS Amide No Alkyl No YesNHS-LC-LC-biotin Primary amine NHS Amide No Alkyl No YesNHS-dPEG4-biotin Primary amine NHS Amide Yes PEO NoNHS-dPEG12-biotin Primary amine NHS Amide Yes PEO NoNHS-iminobiotin Primary amine NHS Amide No None No Yes pH-dependent

avidin bindingNHS-PC-LC-biotin Primary amine NHS Amide No Alkyl Yes Yes Photocleavable

(300–360 nm)Sulfo-NHS-biotin Primary amine Sulfo-NHS Amide Yes None No NoSulfo-NHS-LC-biotin Primary amine Sulfo-NHS Amide Yes Alkyl No NoSulfo-NHS-LC-LC-biotin Primary amine Sulfo-NHS Amide Yes Alkyl No NoSulfo-NHS-SS-biotin Primary amine Sulfo-NHS Amide Yes Alkyl Yes No Cleavable by

reducing agentsPFP-biotin Primary and

secondary aminec)PFP Amide No None No Yes

TFP-PEO-biotin Primary andsecondary amine

TFP Amide Yes PEO No No

Iodoacetyl-LC-biotin Sulfhydryld) Iodoacetyl Thioether No Alkyl No YesIodoacetyl-PEO-biotin Sulfhydryl Iodoacetyl Thioether Yes PEO No NoBMCC-biotin Sulfhydryl Maleimido Thioether No Alkyl No YesMAL-dPEG3-biotin Sulfhydryl Maleimido Thioether Yes PEO No NoHPDB-biotin Sulfhydryl Pyridylthiol Disulfide No Alkyl Yes Cleavable by

reducing agentsBiotin hydrazide Aldehydee) Hydrazide Hydrazone No None No YesBiotin-LC-hydrazide Aldehyde Hydrazide Hydrazone No Alkyl No Yes5-(Biotinamido) pentylamine Carboxyl Amine Amide Yes Alkyl No NoBiotin-PEO-amine Carboxyl Amine Amide Yes PEO No No

a) NHS, N-hydroxysuccinimide; LC, long chain; PEO, polyethylene oxide; PFP, pentafluorophenyl; TFP, tetrafluorophenyl; MAL, mal-eimido; BMCC, 1-Biotinamido-4-(40-[maleimidoethyl-cyclohexane]-carboxamido)butane.

b) e-Amino groups in side chains of lysines; unsubstituted protein N-terminal a-amino groups.c) Same as a); secondary amino groups in arginine and histidine.d) Reduced sulfhydryl groups in cysteines.e) Aldehyde groups can be created by periodate oxidation of carbohydrate residues.

In our hands, this method worked remarkably well forthe elution of molecules containing one biotin residue (e.g.,biotinylated oligonucleotides), but was less efficient whenapplied to the release of heavily biotinylated proteins fromSA-coated resins (unpublished results).

Other approaches to disrupt the biotin–SA complexunder milder conditions have been reported, such as intro-duction of light sensitive biotin phosphoramidites [32] or theuse of polymer conjugates together with SA mutants thatyields temperature or pH dependent release. For example,Ding et al. [47] have conjugated a temperature-sensitivepolymer, poly(N-isopropylacrylamide) (NIPAAm), to agenetically engineered SA to produce a conjugate capable ofbinding biotin at room temperature or lower and releasingbound biotin at 377C. This conjugate can repeatedly bind and

release biotin as the temperature is cycled through the lowercritical solution temperature (LCST) of the polymer. Morerecently, Bulmus et al. [48] conjugated a pH-sensitive poly-mer (a copolymer of NIPAAm and acrylic acid) to the samespecific site on the genetically engineered SA molecule.Lowering the pH was found to cause the polymer to collapseleading to blockage of biotin binding, whereas raising the pHcaused the polymer to fully hydrate thereby permitting biotinto bind.

4 Stability of biotinylated proteins

Investigations in different laboratories [49–55] have shownthat biotin–protein bonds produced by common, commer-



cially available biotinylation reagents may not be stable inbiological fluids. The hydrazone bond formed between bio-tin-LC-hydrazide. and carbohydrates present in proteins isnot stable also in PBS [56]. Care should therefore be takenwhen performing biotinylation reactions in vivo or in thepresence of serum or plasma.

In many of the above-mentioned reports, biotinidaseactivity of biological fluids is reported to catalyze the cleav-age. However, both enzymatic and nonenzymatic mechan-isms have been invoked to account for cleavage of the biotinbond [52]. Free biotin is the primary product released whenbiotin is cleaved from protein in plasma [52]. This findingprovides evidence that the primary site of cleavage is theamide bond between the carboxyl group of biotin and theamino group on the spacer arm (if a spacer arm is present)or the e-amino group of a lysine residue on the protein (ifno spacer arm is present) [52]. Wilbur et al. [50, 54, 57–59]published a series of articles describing modifications of thereagents introducing steric hindrance near the amide bondto protect bond stability. Foulon et al. [53] evaluated twobiotin conjugates in which the amide bond between thevaleric acid side chain of biotin and the prosthetic group ofa small molecule (e.g., 3-iodobenzoate) is reversed (i.e., NH–CO bond); both were stable in serum. Rosebrough’s study[51] indicated that an a-carboxylate in deferoxamine–biotinconjugate could block cleavage in plasma. This conjugatecontained a cysteine linker with a carboxylate alpha to thebiotinamide bond. Bogusiewicz et al. [56] proposed a similarlinker to prevent loss of biotin from labeled antibodies. Thein vitro stability of their biotin-cisteinyl-acetyl-IgG in thepresence of PBS buffer and human plasma was increasedwith respect to all the commercial reagents tested. Authorsinferred that the substituent carboxylate group alpha to thebiotinamide bond sterically hinders the enzymatic cleavageof biotinylated protein. However, given that approximatelyhalf of the cleavage of biotin label occurs by a nonenzymaticmethod, an increased chemical stability conferred by thecarboxyl alpha to the biotinamide bond was also postulated[56].

A NHS-dPEG4-biotin resistant to biotinidase and suit-able for in vivo and clinical studies is now commerciallyavailable from Quanta BioDesign.

5 Metabolic biotinylation of cell surfaceproteins from mammalian cells

An alternative to using synthetic biotinylating reagents forchemical derivatization of proteins in mammalian cells wassuggested by the work of Cronan and coworkers, whodemonstrated that site-specific biotinylation of fusion pro-teins in prokaryotic organisms and S. cervesiae could beachieved. This was accomplished through the use of biotinacceptor domains fused to the target protein of interest [60,61]. By fusing the biotin containing domain of the 1.3S sub-unit of the transcarboxylase of Propionibacterium shermanii

(PSTCD) to a target protein, these researchers demonstratedthat biotinylated proteins could be synthesized in the cyto-plasm of simple organisms. In the prokaryotic P. shermanii,the PSTCD consists of 123 amino acids and is post-transla-tionally biotinylated at the e-amino group of the lysine atposition 89 [62]. The technology could also be applied inmammalian systems, both in cultured cells and in livingmice [63]. In this work, authors demonstrated that the same123 amino acid domain, PSTCD, as well as truncations of thedomain down to 63 amino acids could be used as tags onheterologous proteins to enable their enzymatic biotinylationwithin the cytoplasm and nucleus of living mammalian cells[63]. While the technology allowed proteins in mammaliancells to be readily biotinylated in a site-specific fashion with-out the use of exogenous chemicals, this demonstration wasrestricted to applying the technology to cytoplasmic andnuclear proteins.

In order to accomplish biotinylation of secreted proteinsand cell surface proteins, the same group investigated theuse of BirA, a biotin protein ligase derived from E. coli [64].All eukaryotic and prokaryotic organisms have only one bio-tin ligase [65], which is necessary for the transfer of biotinonto the carboxylases and decarboxylases necessary for thenormal metabolism of the organism. The carboxylasesrequire biotin as a cofactor in order to facilitate carboxyltransfer from one organic molecule to another. Whether thistype of enzyme could be actively synthesized and alsosecreted from mammalian cells was previously unde-termined. Authors determined that the bacterial BirA couldbe expressed and function in the mammalian secretorypathway and found that the primary requirement for meta-bolic biotinylation of secreted proteins routed through themammalian secretion pathway is that a biotin protein ligasemust be cosecreted, since biotin ligase is not normally pres-ent in this cellular compartment. Cosecretion of BirA inmammalian cells with PSTCD-tagged proteins results inrobust biotinylation of the secreted-tagged proteins and ofcell membrane proteins into which the biotin acceptordomains have been incorporated.

Multiple potential applications of this technology exist[64]. One potentially powerful application is the ability topurify PSTCD-tagged proteins in a simple one-step non-denaturing protocol. Using the PSTCD tag, protein fusionscan be easily purified from mammalian cell supernatantsand concentrated, e.g., on monomeric avidin columns, withfew manipulations. This approach has utility for the purifi-cation of secreted mammalian proteins that can only be cor-rectly folded or post-translationally processed in mammaliancells. Another application of this technology is the mem-brane labeling of transfected cells for tagging or purificationpurposes using either monomeric avidin reagents, magneticbeads, or flow cytometry. In this case, no other biotinylatedcell surface protein exists in nature so this novel cell surfacelabel should be specific and have the advantage of compat-ibility with the diverse array of biotin–avidin reagents avail-able. The ability to biotinylate proteins on the cell surface



also opens up the possibility of allowing for the externalattachment of or avidinated drugs, proteins, viruses, or tis-sue-engineering scaffolds to cells expressing biotinylated cellsurface molecules.

6 Proteomic applications

6.1 Quantitative proteomic analysis of membrane

proteins

We have developed a method for the simultaneous recovery,separation, identification and relative quantification of mem-brane proteins [66]. After covalent modification with a cleav-able reactive ester derivative of biotin (Sulfo-NHS-SS-biotin),cells are lysed in the presence of detergents and membraneproteins are purified on SA-coated resin. Contaminant pro-teins can be removed by stringent washing procedures. Bioti-nylated proteins are eluted by reduction of the disulfide bridgecontained in the cleavable linker and tryptically digested. Theresulting peptides are used to generate a 2-D map, based onHPLC separation in the first dimension and mass-spectro-metric analysis in the second dimension (Fig. 5). The use ofinternal peptide standards in MALDI-TOF MS facilitates therelative quantification of peptides (and thus of the corre-sponding protein) [67]. We have exemplified this method bystudying (i) the detection of peptides from a biotinylated BSA-spike, added to an aliquot of human embryonic kidney (HEK)biotinylated membrane proteins before capture on SA-sepharose, with respect to the nonspiked counterpart, and (ii)the relative changes of membrane protein expression of hu-man umbilical chord vein endothelial cells (HUVEC), cul-tured in normoxic or hypoxic conditions [66]. This last studyallowed the recovery of the VE-cadherin/actin/catenin com-plex, revealing an increased accumulation of beta-catenin at2% O2 concentration.

Cell surface protein biotinylation and capture by SA-coated resins led to a substantial increase of the proportionof membrane and membrane-associated proteins identified(up to two-thirds). Enrichment in extracellular matrix com-ponents was also observed. However, we also observed pep-tides corresponding to cytoplasmic proteins. It has beenshown that biotinylation of intracellular proteins is greatlyreduced when using sulfo-NHS-SS-biotin as compared tosulfo-NHS-LC-biotin [25], suggesting that the reducingmilieu of the cytoplasm could be responsible for the cleav-age of the disulfide bond between protein and biotin and asa consequence prevent the isolation of cytoplasmic proteins.It is possible, nonetheless, that cytoplasmic proteins arecopurified by virtue of a strong interaction with membraneproteins and despite of the strong detergents used, or arenot completely eliminated during the washing of the SA-coated resin.

This technique has been successfully applied to proteom-ic characterization of cell surface proteins in vascular endo-thelial cells from the blood and lymph vessel system [68].

6.2 In vivo and ex vivo biotinylation of cell surface

proteins

We have applied some of the above described concepts todevelop a novel methodology for in vivo proteomic discoveryof new targets in cancer, based on the terminal perfusion ofrodents with a reactive ester derivative of biotin, whichenables the covalent modification of proteins that are readilyaccessible from the bloodstream [27]. Biotinylated proteinsfrom total organ extracts can be purified on SA resin in thepresence of strong detergents, digested on-resin and sub-mitted to mass spectrometric analysis for identification.

This in vivo biotinylation procedure led to the identifica-tion of hundreds of proteins in different organs, includingproteins that exhibit a restricted pattern of expression in cer-tain body tissues. Furthermore, the biotinylation of micebearing F9 subcutaneous tumors or orthotopic kidneytumors revealed both quantitative and qualitative differencesin the recovery of biotinylated proteins compared to normaltissues, allowing the identification of accessible markers thatare preferentially expressed in solid tumors [27].

This technique has also been applied to the comparativeproteomic analysis of normal liver and liver metastasis in theF9 model [69] and has been adapted to the first ex vivo prote-omic analysis of human tumor specimens [70]. Using the exvivo perfusion of surgically resected human kidneys withclear cell carcinoma with an active ester derivative of biotinwe were able to identify an unprecedented number of kidneytumor markers. Biotinylated, accessible proteins, mainlylining vascular structures in the normal kidney and in thesolid tumor mass, were purified on SA resin and identifiedusing 2-D peptide mapping and MALDI-TOF/TOF meth-odologies, revealing 637 proteins, 184 of which were onlyfound in tumor specimens. Our methodology opens thepossibility to study human surgical specimens, leading to theidentification of marker proteins that are overexpressedaround vascular structures. Such antigens accessible fromthe vasculature are likely to be suitable targets for ligand-based tumor targeting applications.

It is worth mentioning that the described approach hasbeen applied, with some modifications, to the cell surfacecharacterization of human breast cancer [71] specimens andshould be applicable to several other pathologies (e.g., ather-osclerosis, aneurisms, and chronic inflammatory conditions)as well as to the study of basic physiological andimmunological processes.

7 Conclusions

The avidin–biotin technology has provided the researchcommunity with one of the most powerful tools in bio-chemistry, in particular for protein characterization. Theunique features of this couple of reagents have given scien-tist a handle to grab a particular protein out of a soup ofmany others and manipulate it at their will. The applications



Figure 5. 2-D peptide mapping for quantitative determination of cell surface proteins. Cell surface proteins are covalently modified withsulfo-NHS-SS-biotin, a biotin derivative carrying a cleavable linker and reactive toward primary amino groups. After cell lysis in the pres-ence of detergents, biotin-labeled proteins are purified on SA-coated resin. Following their elution, isolated proteins are proteolyticallydigested in solution. The resulting peptides are fractionated by RP-HPLC and the eluting fractions are automatically premixed with thematrix solution containing internal standard peptides and spotted on MALDI target plates. A 2-D peptide map is established with the dataobtained by MALDI-TOF MS, reporting the HPLC fractions on the y-axis and the m/z ratio on of the measured peptides on the x-axis. Thevalue of the ion current for each ion present in a given fraction is converted by an in-house developed software into bars of different colors,according to a gray-scale. This allows the immediate visualization of the entire experiment and a simplified comparison of closely relatedsamples by matching of corresponding fractions. Signals of interest can be analyzed by MS/MS (up to 15 peaks per fraction), leading topeptide identification.

are countless and have in some cases revolutionized a field,like the ICAT reagents for quantitative proteomics [39].

Desirable future improvements in the technology mightimpart biotinylating reagents a better membrane imperme-ability for characterization of the cell surface protein com-partment and should introduce simplified protocols for therelease of captured proteins when recovery of proteins intheir native, active form is of importance.

I would like to thank Dr. Jascha Rybak and Dr. ChristophRoesli (ETH Zurich) for their invaluable help with the experi-mental work shown in this article and Prof. Michael J. Dunn forcritically reading the manuscript. The financial support of ETHZurich is also gratefully acknowledged.

The author has declared no conflict of interest.

8 References

[1] Boas, M. A., The effect of desiccation upon the nutritiveproperties of egg-white. Biochem. J. 1927, 21, 712–724.

[2] György, P., Rose, C. S., Hofmann, K., Melville, D. B., Du Vig-neaud, V., A further note on the identity of vitamin H withbiotin. Science 1940, 92, 609.

[3] Du Vigneaud, V., Melville, D. B., György, P., Rose, C. S., On theidentity of vitamin H with biotin. Science 1940, 92, 62–63.

[4] György, P., Rose, C. S., Eakin, R. E., Snell, E. E., Williams, R. J.,Egg-white injury as the result of nonabsorption or inactiva-tion of biotin. Science 1941, 93, 477–478.

[5] Pennington, P., Snell, E. E., Eakin, R. E., Crystalline avidin. J.Am. Chem. Soc. 1942, 64, 469.

[6] Wilchek, M., Bayer, E. A., Introduction to avidin-biotin tech-nology. Methods Enzymol. 1990, 184, 5–13.



[7] Green, N. M., Avidin. Adv. Protein Chem. 1975, 29, 85–133.

[8] du Vigneaud, V., Hofmann, K., Melville, D. B., Rachele, J. R.,The preparation of free crystalline biotin. J. Biol. Chem.1941, 140, 763–766.

[9] du Vigneaud, V., Melville, D. B., Folkers, K., Wolf, D. E. et al.,The structure of biotin: A study of desthiobiotin. J. Biol.Chem. 1942, 146, 475–485.

[10] Melville, D. B., Moyer, A. W., Hofmann, K., du Vigneaud, V.,The structure of biotin: The formation of thiophenevalericacid from biotin. J. Biol. Chem. 1942, 146, 487–492.

[11] Harris, S. A., Wolf, D. E., Mozingo, R., Folkers, K., Syntheticbiotin. Science 1943, 97, 447–448.

[12] Trotter, J., Hamilton, J. A., The absolute configuration ofbiotin. Biochemistry 1966, 5, 713–714.

[13] De Clercq, P., Biotin: A timeless challenge for total synthesis.Chem. Rev. 1997, 97, 1755–1792.

[14] Becker, J. M., Wilchek, M., Inactivation by avidin of biotin-modified bacteriophage. Biochim. Biophys. Acta 1972, 264,165–170.

[15] Bayer, E. A., Skutelsky, E., Wilchek, M., The avidin-biotincomplex in affinity cytochemistry. Methods Enzymol. 1979,62, 308–315.

[16] Bayer, E. A., Wilchek, M., The use of the avidin-biotin com-plex as a tool in molecular biology. Methods Biochem. Anal.1980, 26, 1–45.

[17] Bayer, E. A., Wilchek, M., Skutelsky, E., Affinity cytochem-istry: The localization of lectin and antibody receptors onerythrocytes via the avidin-biotin complex. FEBS Lett. 1976,68, 240–244.

[18] Wilchek, M., Bayer, E. A., Biotin-containing reagents. Meth-ods Enzymol. 1990, 184, 123–138.

[19] Bayer, E. A., Viswanatha, T., Wilchek, M., The use of a ho-mologous series of affinity labeling reagents in the study ofthe biotin transport system in yeast cells. FEBS Lett. 1975,60, 309–312.

[20] Orr, G. A., The use of the 2-iminobiotin-avidin interaction forthe selective retrieval of labeled plasma membrane compo-nents. J. Biol. Chem. 1981, 256, 761–766.

[21] Hermanson, G. T., Bioconjugate Techniques, AcademicPress, Inc., San Diego, CA 1996.

[22] Goodman, J. W., in: Stites, M. D., Terr, M. D. (Eds.),BasicHuman Immunology, Appleton & Lange, Los Altos, CA 1991,pp. 101–108.

[23] Buskas, T., Li, Y., Boons, G.-J., The immunogenicity of thetumor-associated antigen Lewisy may be suppressed by abifunctional cross-linker required for coupling to a carrierprotein. Chemistry – A Eur. J. 2004, 10, 3517–3524.

[24] Chen, Y., Chen, C., Wang, Y., Liu-Chen, L.-Y., Ligands reg-ulate cell surface level of the human {kappa} opioid receptorby activation-induced down-regulation and pharmacologi-cal chaperone-mediated enhancement: Differential effectsof nonpeptide and peptide agonists. J. Pharmacol. Exp.Ther. 2006, 319, 765–775.

[25] Peirce, M. J., Wait, R., Begum, S., Saklatvala, J., Cope, A. P.,Expression profiling of lymphocyte plasma membrane pro-teins. Mol. Cell. Proteomics 2004, 3, 56–65.

[26] Porwoll, S., Loch, N., Kannicht, C., Nuck, R. et al., Cell sur-face glycoproteins undergo postbiosynthetic modificationof their N-glycans by stepwise demannosylation. J. Biol.Chem. 1998, 273, 1075–1085.

[27] Rybak, J.-N., Ettorre, A., Kaissling, B., Giavazzi, R. et al., Invivo protein biotinylation for the identification of organ-specific antigens accessible from the vasculature. Nat.Methods 2005, 2, 291–298.

[28] Yu, M.-J., Pisitkun, T., Wang, G., Shen, R.-F., Knepper, M. A.,LC-MS/MS analysis of apical and basolateral plasma mem-branes of rat renal collecting duct cells. Mol. Cell. Proteom-ics 2006, 5, 2131–2145.

[29] Neri, D., Elia, G., A new cleavable biotinylating reagent for

protein derivatiztion. US patent application No. 60/572, 598,2004.

[30] Knorr, R., Trzeciak, A., Bannwarth, W., Gillessen, D., Newcoupling reagents in peptide chemistry. Tetrahedron Lett.1989, 30, 1927–1930.

[31] Bannwarth, W., Knorr, R., Formation of carboxamides withN,N,N0,N0-tetramethyl (succinimido) uronium tetra-fluoroborate in aqueous/organic solvents. Tetrahedron Lett.1991, 32, 1157–1160.

[32] Olejnik, J., Krzymanska-Olejnik, E., Rothschild, K. J., Photo-cleavable biotin phosphoramidite for 50-end-labeling, affini-ty purification and phosphorylation of synthetic oligonu-cleotides. Nucleic Acids Res. 1996, 24, 361–366.

[33] Bayer, E. A., Zalis, M. G., Wilchek, M., 3-(N-Maleimido-pro-pionyl)biocytin: A versatile thiol-specific biotinylatingreagent. Anal. Biochem. 1985, 149, 529–536.

[34] Herforth, C., Heidler, P., Franke, S., Link, A., Polymer-boundreagents for the introduction of spacer-modified biotinlabels. Bioorg. Med. Chem. 2004, 12, 2895–2902.

[35] Visintin, C., Aliev, A. E., Riddall, D., Baker, D. et al., Mem-brane receptor probes: Solid-phase synthesis of biotin-Asp-PEG-arvanil derivatives. Org. Lett. 2005, 7, 1699–1702.

[36] Kahne, T., Ansorge, S., Nonradioactive labelling and immu-noprecipitation analysis of leukocyte surface proteins usingdifferent methods of protein biotinylation. J. Immunol.Methods 1994, 168, 209–218.

[37] Morag, E., Bayer, E. A., Wilchek, M., Immobilized nitro-avi-din and nitro-streptavidin as reusable affinity matrices forapplication in avidin-biotin technology. Anal. Biochem.1996, 243, 257–263.

[38] Ellerbroek, S. M., Wu, Y. I., Overall, C. M., Stack, M. S.,Functional interplay between type I collagen and cell surfacematrix metalloproteinase activity. J. Biol. Chem. 2001, 276,24833–24842.

[39] Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F. et al., Quantita-tive analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 1999, 17, 994–999.

[40] Lee, Y. H., Vacquier, V. D., Reusable cDNA libraries coupledto magnetic beads. Anal. Biochem. 1992, 206, 206–207.

[41] Elgar, G., Schofield, J. P., Carbamoyl phosphate synthetase(CPSase) in the PYR1-3 multigene of Dictyostelium dis-coideum. DNA Seq. 1992, 2, 219–226.

[42] Conrad, F., Krupp, G., A solid phase method for mappingrestriction sites. Nucleic Acids Res. 1992, 20, 6423–6424.

[43] Tong, X., Smith, L. M., Solid-phase method for the purifica-tion of DNA sequencing reactions. Anal. Chem. 1992, 64,2672–2677.

[44] Rybak, J. N., Scheurer, S. B., Neri, D., Elia, G., Purification ofbiotinylated proteins on streptavidin resin: A protocol forquantitative elution. Proteomics 2004, 4, 2296–2299.



[45] Holmberg, A., Blomstergren, A., Nord, O., Lukacs, M. et al.,The biotin-streptavidin interaction can be reversibly brokenusing water at elevated temperatures. Electrophoresis 2005,26, 501–510.

[46] Hyre, D. E., Amon, L. M., Penzotti, J. E., Le Trong, I. et al.,Early mechanistic events in biotin dissociation from strep-tavidin. Nat. Struct. Biol. 2002, 9, 582–585.

[47] Ding, Z., Long, C. J., Hayashi, Y., Bulmus, E. V. et al., Tem-perature control of biotin binding and release with A strep-tavidin-poly(N-isopropylacrylamide) site-specific conjugate.Bioconjug. Chem. 1999, 10, 395–400.

[48] Bulmus, V., Ding, Z., Long, C. J., Stayton, P. S., Hoffman, A.S., Site-specific polymer-streptavidin bioconjugate for pH-controlled binding and triggered release of biotin. Bio-conjug. Chem. 2000, 11, 78–83.

[49] Bogusiewicz, A., Mock, N. I., Mock, D. M., Instability of thebiotin-protein bond in human plasma. Anal. Biochem. 2004,327, 156–161.

[50] Wilbur, D. S., Hamlin, D. K., Pathare, P. M., Weerawarna, S.A., Biotin reagents for antibody pretargeting. Synthesis,radioiodination, and in vitro evaluation of water soluble,biotinidase resistant biotin derivatives. Bioconjug. Chem.1997, 8, 572–584.

[51] Rosebrough, S. F., Plasma stability and pharmacokinetics ofradiolabeled deferoxamine-biotin derivatives. J. Pharmacol.Exp. Ther. 1993, 265, 408–415.

[52] Bogusiewicz, A., Mock, N. I., Mock, D. M., Release of biotinfrom biotinylated proteins occurs enzymatically and none-nzymatically in human plasma. Anal. Biochem. 2004, 331,260–266.

[53] Foulon, C. F., Alston, K. L., Zalutsky, M. R., Synthesis andpreliminary biological evaluation of (3-iodobenzoyl)norbio-tinamide and ((5-iodo-3-pyridinyl)carbonyl)norbiotinamide:Two radioiodinated biotin conjugates with improved stabil-ity. Bioconjug. Chem. 1997, 8, 179–186.

[54] Wilbur, D. S., Hamlin, D. K., Chyan, M. K., Kegley, B. B.,Pathare, P. M., Biotin reagents for antibody pretargeting. 5.Additional studies of biotin conjugate design to providebiotinidase stability. Bioconjug. Chem. 2001, 12, 616–623.

[55] Sabatino, G., Chinol, M., Paganelli, G., Papi, S. et al., A newbiotin derivative-DOTA conjugate as a candidate for pre-targeted diagnosis and therapy of tumors. J. Med. Chem.2003, 46, 3170–3173.

[56] Bogusiewicz, A., Mock, N. I., Mock, D. M., A biotin-proteinbond with stability in plasma. Anal. Biochem. 2005, 337, 98–102.

[57] Wilbur, D. S., Chyan, M. K., Pathare, P. M., Hamlin, D. K. et al.,Biotin reagents for antibody pretargeting. 4. Selection ofbiotin conjugates for in vivo application based on their dis-sociation rate from avidin and streptavidin. Bioconjug.Chem. 2000, 11, 569–583.

[58] Wilbur, D. S., Pathare, P. M., Hamlin, D. K., Buhler, K. R.,Vessella, R. L., Biotin reagents for antibody pretargeting. 3.Synthesis, radioiodination, and evaluation of biotinylatedstarburst dendrimers. Bioconjug. Chem. 1998, 9, 813–825.

[59] Wilbur, D. S., Pathare, P. M., Hamlin, D. K., Frownfelter, M. B.et al., Evaluation of biotin-dye conjugates for use in an HPLCassay to assess relative binding of biotin derivatives withavidin and streptavidin. Bioconjug. Chem. 2000, 11, 584–598.

[60] Cronan, J. E., Jr., Biotination of proteins in vivo. A post-translational modification to label, purify, and study pro-teins. J. Biol. Chem. 1990, 265, 10327–10333.

[61] Reed, K. E., Cronan, J. E., Jr., Escherichia coli exports pre-viously folded and biotinated protein domains. J. Biol.Chem. 1991, 266, 11425–11428.

[62] Jank, M. M., Bokorny, S., Rohm, K., Berger, S., Expressionand biotinylation of a mutant of the transcarboxylase carrierprotein from Propioni shermanii. Protein Expr. Purif. 1999,17, 123–127.

[63] Parrott, M. B., Barry, M. A., Metabolic biotinylation ofrecombinant proteins in mammalian cells and in mice. Mol.Ther. 2000, 1, 96–104.

[64] Parrott, M. B., Barry, M. A., Metabolic biotinylation ofsecreted and cell surface proteins from mammalian cells.Biochem. Biophys. Res. Commun. 2001, 281, 993–1000.

[65] Chapman-Smith, A., Cronan, J. E., Jr., In vivo enzymaticprotein biotinylation. Biomol. Eng. 1999, 16, 119–125.

[66] Scheurer, S. B., Rybak, J. N., Roesli, C., Brunisholz, R. A. etal., Identification and relative quantification of membraneproteins by surface biotinylation and two-dimensional pep-tide mapping. Proteomics 2005, 5, 2718–2728.

[67] Nelson, R. W., McLean, M. A., Hutchens, T., Quantitativedetermination of proteins by matrix-assisted laser desorp-tion/ionization time-of-flight mass spectrometry. Anal. Bio-chem. 1994, 66, 1408–1415.

[68] Roesli, C., Mumprecht, V., Neri, D., Detmar, M., Identificationof the surface-accessible, lineage-specific vascular pro-teome by two-dimensional peptide mapping. FASEB J.2008, fj.07–100529.

[69] Rybak, J.-N., Roesli, C., Kaspar, M., Villa, A., Neri, D., Theextra-domain A of fibronectin is a vascular marker of solidtumors and metastases. Cancer Res. 2007, 67, 10948–10957.

[70] Castronovo, V., Waltregny, D., Kischel, P., Roesli, C. et al., Achemical proteomics approach for the identification ofaccessible antigens expressed in human kidney cancer. Mol.Cell. Proteomics 2006, 5, 2083–2091.

[71] Castronovo, V., Kischel, P., Guillonneau, F., de Leval, L. et al.,Identification of specific reachable molecular targets in hu-man breast cancer using a versatile ex vivo proteomicmethod. Proteomics 2007, 7, 1188–1196.


biotinylation reagents for the study of cell surface proteins

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