R EVIEW
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 most commonly exploited tools in chemistry and biology.Methods for derivatization with biotin of a variety of molecules(in particular,proteins)have been introduced,in order to allow their efficient recovery,immobilization and detection with avidin-based reagents.The field has evolved very rapidly and the applications have become more and more sophisticated.Cell surface protein studies have enormously benefited from refinements of this technology.It is now possible to specifically biotinylate one single membrane protein or to fish out a membrane receptor bound to its ligand.The release of biotinylated molecules from the avidin-based reagents,however,may still represent a major problem,due to the stability of the complex.This review will examine the biotin–avidin technology for the study of cell surface proteins,discussing reagents and tech-niques as well as examples of applica
tions in quantitative proteomics.Received:February1,2008 Revised:April1,2008 Accepted:April16,2008
Keywords:
Avidin/Biotin/Biotinylation/Cell Surface Proteins
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1Introduction
In1927,Margaret A.Boas,a research fellow at the Depart-ment of Experimental Pathology of the Lister Institute in London,observed that rats fed large quantities of raw egg white developed severe dermatitis,indicating malnutrition which eventually caused death[1].Vitamin H,whose prop-erties and structure were later found identical to those of biotin[2,3],prevented such dermatitis.The malnutrition was eventually attributed to the depletion of biotin by an unknown factor,proteinaceous in nature,to which the name of avidalbumin was initially given[3].This protein present in consistent amount in the egg white was able to form a very stable complex with the vitamin and interfered with the ani-mal’s nutrition.
Already in1941,the extraordinary affinity of the protein, renamed avidin,for biotin was recognized[4].In1942,crys-tallization of avidin in a pure form was accomplished[5]and it became rapidly clear that the interaction of the relatively small vitamin H molecule with the egg-white glycoprotein avidin and with the related bacterial protein streptavidin(SA)[6] could be easily converted in a affinity-based tool for several different purposes.Since then,and for the following decades, chemical modification of a variety of molecules with biotin has 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 SA ,fluorophore-,horseradish peroxidase-,or alkaline phosphatase-conjugates),or efficiently captured on avidin/SA-coated solid ,resins,magnetic beads, microtiter plates,chips).However,due to the very high stabil-ity of the biotin–SA complex(K d,10215M)[7],the elution of biotinylated molecules from SA-coated surfaces has repre-sented a real challenge for many years.
This article will review the biotin–avidin technology for the study of cell surface proteins,providing examples of both commercially available and laboratory-developed reagents,as well as techniques for elution of biotinylated proteins from avidin and SA.Alternative metabolic techniques for protein
Correspondence:Dr.Giuliano Elia,Mass Spectrometry Resource, Conway Institute of Biomolecular an
d Biomedical Research,Uni-versity College Dublin,Belfield,Dublin4,Republic of Ireland
E-mail:giuliano.elia@ucd.ie
Fax:+353-1-7166703
Abbreviations:DST,disuccinimidyl tartrate;EDC,LC,long chain; NHS,N-hydroxysuccinimide;PEO,polyethylene oxide;PFP,pen-tafluorophenyl;SA,streptavidin;TFP,tetrafluorophenyl
DOI10.1002/pmic.200800097
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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.
2Biotinylating reagents
A chemical reagent to be used for protein biotinylation experiments is made up of the following building blocks:
(i)The biotin moiety,for the subsequent interaction of biotinylated proteins with avidin/SA-based reagents;the valeric acid side chain of the biotin molecule is important in the interaction with avidin,but its terminal carboxylic group can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules.The use of biotin in conjunction with SA yields the highest binding affinity and allows the use of strong detergents during the purifica-tion of relatively insoluble proteins.
(ii)A spacer of sufficient length to allow protein capture by immobilized SA.The spacer is possibly the most impor-tant variable,in that it largely determines the , solubility)of the biotinylating reagent and of the derivatized proteins.The spacer can also be cleavable by chemical or physical agents to facilitate protein release after capture.
(iii)A reactive moiety for the covalent binding of biotin to the protein(s).The most common reactive groups employed include reactive esters,like the N-hydroxysuccinimide(NHS) group,which undergoes a nucleophilic substitution reaction in the presence of primary ,e-amino group in exposed lysine residues in proteins),or reactive imides,for instance the maleimido group,which reacts with free thiol groups(like the ones contained in cysteine residues of proteins).
The importance of these three different building blocks is discussed in the following section.
2.1Biotin,biotin derivatives,and their synthesis The empirical formula of biotin was determined by du Vig-neaud et al.in1941[8]and its structure was established by the same group in1942[9,10].This structure was confirmed by the first total synthesis of racemic biotin in the Merck Re-search Laboratories[11].Finally,in1966,X-ray crystal-lographic analysis established the absolute configuration of natural(1)-biotin as1(Fig.1)[12].To date,more than40 different 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 conjugate biotin to other chemical groups,taking advantage of the
free Figure1.Absolute configuration of natural(1)-inal carboxylic group.In initial studies in which the concepts for biotinylating proteins were first laid down,bio-tinyl-NHS ester(NHS-biotin)was used for incorporating the biotin moiety via lysines into several bacteriophages,lectins, and antibodies[14–17].NHS-biotin was obtained by direct coupling of biotin and NHS in the presence of dicyclohex-ylcarbodiimide in dimethylformamide(DMF)[18].NHS-biotin has been for long the most po
pular biotinylating reagent and has been used almost exclusively for biotinyla-tion of proteins.Reasons for its widespread usage are that lysine residues are numerous in most proteins and that they frequently occupy an accessible position.Furthermore,the lysine residues are usually not directly involved in proteins0 biological activity,and their modification generally has little effect on the interaction of a protein with its substrate.The utility and efficiency of action of this reagent are underlined by the dozens of commercial enterprises(Pierce,Molecular Probes,Invitrogen,Quanta BioDesign,to name but a few) that include NHS-biotin in their catalogs.
In recent years NHS-biotin has been replaced with longer-chain homologs, e.g.,biotinyl-e-aminocaproyl-NHS (NHS-long chain(LC)-biotin)ester,in order to overcome steric hindrance problems and ameliorate the accessibility of the biotin residue to avidin/SA-based reagents.Preparations of NHS-LC-biotin start from NHS-biotin in DMF,which is reacted with bicarbonate solution of e-aminocaproic acid. The crystallized biotinyl-e-aminocaproic acid is then reacted with NHS as described above for NHS-biotin.
Water-soluble ,N-hydroxysulfosuccinimide derivatives 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 ,biotinyl-p-nitrophenyl ester(BNP)and its homologs)are also efficient reagents for i
ncorporating biotin into proteins via amino groups of lysine[19].
In1981,Orr[20]introduced the use of2-iminobiotin, the cyclic guanidino analog of biotin,in cell surface protein studies making use of immobilized avidin to recover the labeled components,uncontaminated by other cytosolic and membrane components.The pH-dependent interac-tion of2-iminobiotin with avidin facilitates recovery.At high pH,the free base form of2-iminobiotin retains the high affinity specific binding to avidin characteristic of biotin,whereas at acidic pH values,the salt form of the analog interacts poorly with avidin.
Iminobiotin-containing biotinylating reagents are commercially ,from Pierce and Perkin-Elmer)but are less widespread than their biotin-con-taining counterparts,also due to the reduced tolerance to detergents of the avidin–iminobiotin complex[20].
2.2Spacer groups
At the end of the1980s it became apparent that,for steric hindrance reasons,interactions between avidin and a bioti-nylated protein could be dramatically improved by increasing
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the length of the spacer arm that connects biotin to the bulk of the biomolecule.
Sulfo-NHS-LC-biotin mentioned above(spacer arm length8.8Å)is probably the most popular biotinylation reagent currently available on the market.To increase spacer length,Pierce(and other companies)proposes also a Sulfo-NHS-LC-LC-biotin(spacer arm20.4Å),in which two ali-phatic aminocaproic acid chains are arranged head-to-tail between the biotin and the sulfo-NHS group.Although these molecules are soluble in water,the nature of the LC linker is substantially hydrophobic.Once labeled,the LC-biotin will seek hydrophobic regions in the protein and hide in them, making it less available to avidin.At the same time,the hydrophobic LC-biotin labels may cause serious agglutina-tion and precipitation problems.
Monodisperse PEG-based spacer arms are now available from different ,Pierce,EZ-Link PEO-biotin; Quanta BioDesign,discrete PEG™;KPL,Surelink™).Mono-disperse PEG compounds and their derivatives are suitable for a wide range of therapeutic,diagnostic,and molecular engineering applications.Spacer lengths of up to46Åhave been introduced for labeling and capture of residues deeply buried in folded protein regions and are commercially avail-able(Quanta).
PEG-based spacers are preferred substitutes to aliphatic methylene chain spacers in that these linke
rs are extremely water soluble and hydrophilic.In addition to being water soluble,the PEG linkers are also soluble in organic sol-vents,like DMF and DMAC,but especially in methylene chloride.
Agglutination data have compared the Sulfo-NHS-LC-biotin with NHS-dPEG4-biotin(Quanta),which has the dPEG4spacer(comparable in length to Sulfo-NHS-LC-LC-biotin).The data shows that human IgG biotinylated with the sulfo-NHS-LC biotin precipitates within a couple of weeks, while human IgG biotinylated with NHS-dPEG4-biotin shows no agglutination on the third week[21].
It should also be mentioned that PEG-based spacers have been shown to be less immunogenic than aliphatic chain-based ones in in vivo applications[22].Using a carrier protein conjugated to a peptide by a linker is a widespread approach to elicit a peptide-specific immune response.However,the use of conventional linkers contributes to generate a linker-dependent response.Alkyl linkers containing more than two or three methylene groups are highly immunogenic[23], sometimes requiring the alkyl spacer length to be shortened to reduce this undesirable property.In contrast,PEG is well known to be nonimmunogenic and in the above sense dPEG-based linkers are nonimmunogenic as well[21].
Cleavable spacers have been introduced and made commercially available with the aim of facilitating the release 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 like b-mercaptoethanol or DTT.Reagents containing this cleavable group come associated to both aliphatic chain-based and PEG-based ,in Sulfo-NHS-SS-biotin or in NHS-SS-dPEG4-biotin)and have been used in a number of studies of cell surface proteins[24–26].Owing to the nature of the cleavable linker,care should be taken when using these reagents in a reducing environment. Our own experience has shown for instance that these reagents are not compatible with in vivo biotinylation procedures[27].The reducing intracellular milieu is also not adapted to the use of disulfide bridge-containing reagents.This fact can however be turned into an advan-tage when dealing with cell surface protein studies.In fact,notwithstanding the presence of a charged group that should render it membrane-impermeable,sulfo-NHS-LC-biotin has been shown to be able to permeate biological membranes,leading to sample contamination by cytoplasmic proteins[28].The use of a cleavable linker in these conditions limits contamination,due to intracel-lular cleavage of the reagent[25].
We have proposed a cleavable linker based on a vicinal diol group,which can be cleaved by mild treatment with oxidants like sodium metaperiodate[29].It is possible to chemically synthesize this reagent with traditional organic synthesis methods[30,31],as depicted in Fig.2.Alter-natively,a rapid way of producing the same biotinylating ,to label lysine residues of proteins and con-taini
ng a vicinal diol in the linker region,is to combine the use of an homobifunctional crosslinker(a NHS diester of tartaric acid)with a biotin derivative containing a terminal primary amine group(Fig.3).The reaction proceeds in three steps.First,N-(3-aminopropyl)-biotinamide tri-fluoroacetate(Fluka,Buchs,Switzerland)is dissolved in PBS pH7.8and disuccinimidyl tartrate(DST)(Genotech, Lausanne,Switzerland)is dissolved in the same buffer, containing20%DMSO.The two substances are mixed in a stoichiometric ratio of1.2:1equivalents and allowed to react at RT for15min to form the intermediate7(Fig.3).The reaction mixture is then added to the protein-containing sample in PBS pH7.8and biotinylation allowed to take place at RT for30min.Excess unreacted7and DST are quenched with an excess of a primary amine-containing reagent(50mM Tris-Cl pH8.0).
As a proof-of-principle experiment,we biotinylated an aqueous solution of BSA and released the biotinylated pro-tein,after capture on a SA-sepharose column,by means of a dilute solution of an oxidant,like sodium metaperiodate (Fig.4).This new reagent allows investigators to biotinylate proteins in a wide range of environmental conditions, including reducing ,the intracellular milieu)and to release biotinylated proteins,captured on a avidin resins,simply by treatment with dilute solutions of oxidants.
Photocleavable linkers are also commercially available. Pierce has introduced NHS-PC-LC-biotin as a unique amine reactive,photocleavable biotin analog with an extended spacer arm.The spacer arm imparted by NHS-PC-LC-biotin is approximately31.4Åin length.A photocleavable1–2
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Figure2.Organic synthesis of a NHS-LC-biotin derivative containing a vicinal diol group as cleavable linker.Tartaric acid2is reacted with N,N,N0,N0-tetramethyl(N-hydroxysuccinimido)uronium tetrafluoroborate3to yield a mixture of esters of tartaric acid,the N,N,N0,N0-tetra-methyl uronium hemitartrate4(IUPAC name:[(3-carboxy-2,3-dihydroxy-propionyloxy)-dimethylamino-methylene]-dimethyl-ammonium) and the succinimidyl hemitartrate5(IUPAC name:2,3-Dihydroxy-succinic acid mono-(2,5-dioxo-pyrrolidin-1-yl)ester).These esters can then be reacted with a primary amine-containing biotin derivative(N-(3-aminopropyl)-biotinamide trifluoroacetate6)to yield the biotin-amidopropyl hemitartramide7(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 of7can 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 esters8(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 and1-(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 for reaction with lysyl residues in proteins.
(nitrophenyl)-ethyl moiety is attached to the biotin through this long spacer arm,separating the biotin from the amine reactive NHS group at the end of the molecule.The con-jugate formed with NHS-PC-LC-biotin undergoes an effi-cient photocleavage upon illumination with300–360nm light[32],resulting in the rapid release of the target protein in an unmodified form.Photocleavable biotin derivatives are a useful alternative to chemically cleavable ones;yet,in some experimental conditions,it is not easy to achieve efficient illumination of the sample.
ignore subsequent bad blocksOther types of linkers have been proposed in the litera-ture.Biocytin(N-e-biotinyl-L-lysine)is a naturally occurring breakdown product of biotin-requiring enzymes.Synthesis of biocytin derivatives containing a propionyl group for increasing the spacer length has been described[33].Her-forth et al.[34]introduced an on-bead construction protocol which permits the simple preparation of biotin labels with custom tailored spacers using established amide bond forming procedures.On bead synthesis leads to simple product isolation because the excess of polymer-bound reagent that is applied to drive reactions to completion can be removed by filtration.This allows for the fast and con-venient access to different biotinylated compounds in high yield and purity.
Solid-phase synthesis of biotin-PEG derivatives is also at the basis of a new method for the preparation of crosslinking probes for membrane receptors[35].The method was suc-cessfully applie
d to development of a biotin-Asp-PEG-arvanil probe for the study of cannabinoid receptors,and holds pro-mise of becoming a general method for identification of receptors 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)have also been made available by several companies.These reagents provide an easy method for labeling proteins and accurately measure total biotin incorporation with a single step.The number of biotins can be quantitated by spectro-photometric analysis at UV wavelengths.
2.3Reactive groups for protein derivatization
The chemical groups that have most commonly been tar-geted for protein derivatization are the primary amino
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Figure3.Alternative generation of a vicinal diol-containing biotinylating reagent.The biotin derivative6(N-(3-aminopropyl)-biotinamide trifluoroacetate),dissolved in an aqueous buffer,is reacted
with the reactive diester9DST or disulfosuccinimidyl tartrate(DSST).The stoichiometric ratio of the two reagents is adjusted in order to maximize the yield of the product biotinamide(sulfo)succinimidyl tartrate8 (biotin-(S)ST)and to prevent,to the largest extent possible,crosslinking of protein molecules.The reaction product can be ,by HPLC)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 for30260min and the exposed lysyl residues of the protein(s)are bound to the vicinal diol-containing biotin derivative.Finally,the excess unreacted biotin derivatives is quenched with an excess of a primary amine-containing buffer solution (e.g.,Tris-Cl50mM pH8.0)and all low molecular weight contaminants are removed by gel filtration chromatography(PD-10columns).
groups,which are abundant in the vast majority of proteins in the form of lysine side chain e-amines and N-terminal a-amines.Amine-reactive biotinylation reagents can be divided into two groups,according to their solubility in aqueous Solutions.NHS-esters of biotin are essentially water insoluble.For reactions in aqueous solution,they must first be dissolved in DMSO and DMF,which are compatible with most proteins at20%final concentration.The organic sol-vent forms an emulsion in the aqueous phase,allowing the biotinylation reaction to proceed.These hydrophobic NHS-esters of biotin are therefore also membrane permeable.
Sulfo-NHS-esters of biotin are soluble in water up to10 mM.They are prone to hydrolysis in aqueous milieu and should be dissolved just before their use.The water solubility of sulfo-NHS-esters is imparted to the compound by the sulfonate 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 used as cell surface biotinylation reagents.
Alternatively,primary and secondary amino groups in proteins can be targeted by pentafluorophenyl-or tetra-fluorophenyl ester derivatives of PFP-biotin and TFP-PEO-biotin,Pierce).These molecules are more reactive towards amino groups at a slightly basic pH and are less prone to hydrolysis.
Another very common target for protein derivatization is the free sulfhydryl group,found in cysteine residues.Three different reactive groups can be employed to target sulfhy-dryl groups for biotinylation.The most specific method uses reactive maleimide groups,which are extremely reactive to-ward free sulfhydryls at pH7.The reaction of maleimide with free thiols is carried out at pH6.5–7.5because at higher pH 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 sulfhydryls is the iodoacetyl moiety.Iodoacetyl-LC-Biotin is not water soluble and must be dissolved in a solvent before use in an aqueous reaction mixture.The iodoacetyl group mainly reacts with cysteine thiol groups at pH7.5–8.5,resulting in a stable thioether bond.At this pH,cross-reactivity with amine,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 to6.9–7.0.Incuba-tion time must in this case be increased(up to a week).His-tidyl side chains may also take part in reactions above pH5.0.
Finally,pyridyldithiol groups are also used to derivatize free sulfhydryls with biotin by a mechanism of disulfide exchange,which results in the formation of a mixed disulfide bond.The reaction of HPDP-biotin((N-[6-(biotinamido)-
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