A Simple and Effective Cleavable Linker for Chemical Proteomics Applications

Abstract

The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion. The study of metabolically labeled or probe-modified proteins is an important area in chemical proteomics. Isolation and purification of the protein targets is a necessary step before MS identification. The biotin-streptavidin system is widely used in this process, but the harsh denaturing conditions also release natively biotinylated proteins and non-selectively bound proteins. A cleavable linker strategy is a promising approach for solving this problem. Though several cleavable linkers have been developed and tested, an efficient, easily synthesized, and inexpensive cleavable linker is a desirable addition to the proteomics toolbox. Here, we describe the chemical proteomics application of a vicinal diol cleavable linker. Through easy-to-handle chemistry we incorporate this linker into an activity-based probe and a biotin alkyne tag amenable for bioorthogonal ligation. With these reagents, background protein identifications are significantly reduced relative to standard on-bead digestion. The covalent modification of proteins by small molecules within a complex proteome is a major theme in chemical biology and proteomics. An effective method for the detection of posttranslational modifications of proteins is the metabolic incorporation of modified biomolecules such as tagged carbohydrates or lipids (1Heal W.P. Tate E.W. Getting a chemical handle on protein post-translational modification.Org. Biomol. Chem. 2010; 8: 731-738Crossref PubMed Scopus (41) Google Scholar). Reversible interactions of enzyme inhibitors, natural products, or drugs can be detected when one appends photocrosslinking agents, thereby facilitating target discovery (2Dubinsky L. Krom B.P. Meijler M.M. Diazirine based photoaffinity labeling.Bioorg. Med. Chem. 2012; 20: 554-570Crossref PubMed Scopus (274) Google Scholar, 3Geurink P.P. Prely L.M. van der Marel G.A. Bischoff R. Overkleeft H.S. Photoaffinity labeling in activity-based protein profiling.Top. Curr. Chem. 2011; 324: 85-113Crossref Scopus (89) Google Scholar). A particularly interesting example of protein labeling is activity-based protein profiling (ABPP) 1The abbreviations used are:ABPPactivity-based protein profilingABPactivity-based probeCatcathepsinDIEAN,N-diisopropyl-N-ethylamineDMEMDulbecco's modified Eagle mediumFmocN-(9-fluorenyl)methoxycarbonylHBTU2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateMHCmajor histocompatibility complexSBsample bufferSPPSsolid phase peptide synthesis. 1The abbreviations used are:ABPPactivity-based protein profilingABPactivity-based probeCatcathepsinDIEAN,N-diisopropyl-N-ethylamineDMEMDulbecco's modified Eagle mediumFmocN-(9-fluorenyl)methoxycarbonylHBTU2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphateMHCmajor histocompatibility complexSBsample bufferSPPSsolid phase peptide synthesis. (4Cravatt B.F. Wright A.T. Kozarich J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.Annu. Rev. Biochem. 2008; 77: 383-414Crossref PubMed Scopus (879) Google Scholar, 5Heal W.P. Dang T.H.T. Tate E.W. Activity-based probes: discovering new biology and new drug targets.Chem. Soc. Rev. 2011; 40: 246-257Crossref PubMed Scopus (146) Google Scholar), which utilizes the intrinsic catalytic activity of a target enzyme for the covalent attachment of an affinity or visualization tag. ABPP makes use of small molecules (activity-based probes (ABPs)) that react with the active form of a specific enzyme or enzyme class by means of a “warhead,” which is often derived from a mechanism-based enzyme inhibitor (Fig. 1A). DCG-04, for example, is based on the naturally occurring inhibitor E-64 and targets the papain family of cysteine proteases via covalent attachment of the epoxysuccinate group to the active site cysteine (Fig. 1B) (6Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). activity-based protein profiling activity-based probe cathepsin N,N-diisopropyl-N-ethylamine Dulbecco's modified Eagle medium N-(9-fluorenyl)methoxycarbonyl 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate major histocompatibility complex sample buffer solid phase peptide synthesis. activity-based protein profiling activity-based probe cathepsin N,N-diisopropyl-N-ethylamine Dulbecco's modified Eagle medium N-(9-fluorenyl)methoxycarbonyl 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate major histocompatibility complex sample buffer solid phase peptide synthesis. Bulky fluorophore or biotin tags on chemical probes might interfere with efficient protein binding. Moreover, they can negatively influence the cell permeability of probes, which therefore limits their applicability in in vitro experiments. Bioorthogonal chemistries, such as the Bertozzi-Staudinger ligation (7Saxon E. Bertozzi C.R. Cell surface engineering by a modified Staudinger reaction.Science. 2000; 287: 2007-2010Crossref PubMed Scopus (1837) Google Scholar) and the 1,3-bipolar cycloaddition of an azide and an alkyne (click chemistry) (8Kolb H.C. Finn M.G. Sharpless K.B. Click chemistry: diverse chemical function from a few good reactions.Angew. Chem. Int. Ed. 2001; 40: 2004-2021Crossref PubMed Scopus (11474) Google Scholar), allow tandem labeling strategies in which a biotin or a fluorophore is attached to an enzyme probe complex in a separate step. Consequently, the probes themselves only carry azide or alkyne groups as “mini-tags.” Tandem labeling using bioorthogonal chemistry has now become a widely used strategy to label biomolecules in lysates and in live cells (9Prescher J.A. Bertozzi C.R. Chemistry in living systems.Nat. Chem. 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The quantitative elution of biotinylated proteins requires harsh conditions (12Rybak J.N. Scheurer S.B. Neri D. Elia G. Purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution.Proteomics. 2004; 4: 2296-2299Crossref PubMed Scopus (100) Google Scholar), which lead to contamination of the sample by endogenous biotinylated and non-specifically bound proteins. These other proteins will be identified together with the real protein targets. Given that subsequent target validation with secondary assays can be a costly and time-consuming process, a reduction in false positive identifications is highly desirable. For cleaner protein identification, cleavable linker strategies (13Leriche G. Chisholm L. Wagner A. Cleavable linkers in chemical biology.Bioorg. Med. Chem. 2012; 20: 571-582Crossref PubMed Scopus (155) Google Scholar) that allow the selective release of target proteins have been developed (Fig. 1C). 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Overkleeft H.S. A cleavable linker based on the for activity-based protein Chem. Int. Ed. 2010; 49: PubMed Scopus Google Scholar). The of of these linkers is or to which limits their general application in chemical proteomics. a cleavable linker is stable under a variety of conditions, is and and can be in a of chemical to these by using a vicinal diol as a cleavable linker vicinal are with the is cleaved (Fig. of proteins can result in such as the cleavage of carbohydrates or the of and these in proteins and are therefore of the conditions of cleavage are with proteins. This has been in the for example, by application in the detection of interactions D. F. of with 17: PubMed Scopus Google Scholar) and the of class molecules M. A. H. major histocompatibility by a Am. Chem. Soc. 2009; PubMed Scopus Google Scholar). this we the chemical proteomics application of diol cleavable linker that the of the linker and probe is that the linker is with tandem click that enrichment and release of probe targets is efficient, and that the of targets with significantly background in on-bead from and groups via with and a catalytic of The by means of and as a light in one with one of via as a in of with and cleavable under the influence of followed by with which the of the under of an with and (6Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). cleaved from the and via to with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and the with an of in The and to biotin under the influence of and in The and the in for of the The and the via The of the linker and probes and their are in the For the a of into small and with buffer and The on the for to and for to cell The in and The protein via the or protein cells in and and in a with For in cells with probes with a of cells with with or via the use of a cell and by means of Cell with and with in buffer or in buffer and for The for to cells and cell The in and via or protein with or for an labeled and with The probes in the by The with streptavidin for and to the The with and into with The other with in buffer in the for and the the with and buffer and for of sample a and proteins via streptavidin For the detection of cleavage by of in buffer with for probe via a and the with streptavidin for The streptavidin with buffer and the with buffer labeled by the probe from the by with or by in buffer in the the cleavage the chemical the with and the with and to the cleavage of sample on a and the proteins by means of to the of the cleavage specific chemical cleavage or release by the with and for streptavidin an labeled proteome in and and the for to The by or by a with for probe via a and the with streptavidin for from the via The with a of buffers in in in and in and the with buffer used for on-bead or chemical For on-bead streptavidin with bound proteins in of buffer proteins reduced in the of for by the addition of and for in the by the addition of The reduced by the addition of with and on a elution via the addition of elution buffer in for in the The cleavage buffer and with The and with a the and the sample for analysis as for the on-bead digestion. For in vitro proteome the probe of to cell in and for probe via a and the to with click probe via a and the with streptavidin for and chemical release followed by to the protocol For in proteome cells labeled in for with for and with and a using a buffer labeled protein used for the of on-bead and chemical release the protocol for in vitro proteome of by an to an from the with and for to a with a of in buffer A in of and to an and using a from to of buffer in a of The mass in MS and Full MS in the and tandem MS using the in Tandem mass for to peptide using A. S. M. for peptide modification 2007; 4: PubMed Scopus Google Scholar). from for and and and of MS using the and to for peptide and protein identification. The the with to for the proteins or the proteome of 2010; proteins for the cell including protein of and with the a of and a of a of and for the of the of cysteine the of cysteine and the modification by cleaved with and and or and cleaved with and and as The result using of peptide highly and in a peptide and protein false discovery of to as a vicinal cleavable linker. is small relative to other cleavable linkers and is therefore to have influence on probe might the of probes in buffers and interactions with of proteins M. A. A. and of for the reduction of binding proteins on affinity Med. Chem. 2004; PubMed Scopus Google Scholar). it is stable under and reducing conditions, and it is therefore with buffer used in as a for incorporation into chemical probes, it can be on and in good from inexpensive in group (Fig. can be used in and solid phase synthesis. we a solid of which is a cleavable of For tandem labeling of azide probes, we the alkyne cleavable biotin reagent which in in (Fig. the diol cleavable linker within the of a probe be cleaved by this we with and identified the of the cleavage of the which is the cleavage as well as the of the to for reactions with proteins and we also cleavage in the of a which result in reactions this we the and release of proteases from a a of the cathepsin with the a which a variety of identified D. A. L. Burlingame A. Medzihradszky K.F. Bogyo M. for the Cell. Proteomics. 2002; 1: Full Text Full Text PDF PubMed Scopus Google Scholar), probes a activity-based labeling that the cleavable linker influence the and of the The labeled cathepsin proteases also from the proteome via with with to release of the but on (Fig. cleavage of the diol linker in of the biotin used for detection, cleaved proteins are by means of streptavidin therefore also in to the selective release or release in sample an to the cleavage conditions, we target proteins from the streptavidin using and Although to release from class molecules M. A. H. major histocompatibility by a Am. Chem. Soc. 2009; PubMed Scopus Google Scholar), this or result in the efficient cleavage of proteins from streptavidin with the cathepsin as well as proteins of and (Fig. These proteins also in the a of with a of non-selectively bound detected in that proteins bound to the streptavidin of the sample release the of the are on the and the N. and modification of and 2010; PubMed Google Scholar), and their will also be the we the and the streptavidin the of the cleaved from chemical cleavage and release that this has influence on the of the elution (Fig. that the cleavage conditions and to of the the also via streptavidin (Fig. biotinylated proteins detected in the that the cleavage the of the and the biotin Although biotinylated are tools for the study of in cell and they have applicability in and in of their cell Tandem labeling in ABPP in by the groups of used click chemistry for the detection of A.E. Cravatt B.F. enzyme activities in using click chemistry Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A.E. Cravatt B.F. Activity-based protein profiling in using a azide-alkyne cycloaddition.J. Am. Chem. Soc. PubMed Scopus Google Scholar), and a Bertozzi-Staudinger ligation for labeling of in live cells H. van E. van van der Marel G.A. Overkleeft H.S. Chemistry in living detection of active by a labeling Chem. Int. Ed. PubMed Scopus Google Scholar). used the cathepsin H.C. J. E. van der R. probe for the analysis of cathepsin cysteine proteases in living Chem. Biol. 1: PubMed Scopus Google Scholar) to that the diol cleavable linker is with tandem labeling and can be used to targets in live cells of the cell with for active detected as by streptavidin cell and with the cathepsin probe (Fig. This that is cell and with the of click chemistry with alkyne tag cathepsin and (Fig. of these can be by the inhibitor that the labeling in an activity-dependent click chemistry detection can also be on cell lysates (Fig. we that protein targets in in vitro and to that the cleavable linker can protein in protein by we release with on-bead a used method for the of proteins. A proteome with and to proteins. The streptavidin into and to an on-bead or a release followed by a digestion. analysis the of in which is in D. A. L. Burlingame A. Medzihradszky K.F. Bogyo M. for the Cell. Proteomics. 2002; 1: Full Text Full Text PDF PubMed Scopus Google Scholar). The cleavage conditions are to the identified the target we in the sample from chemical cleavage in that via on-bead this have an influence on the of the proteins such as and are in cell and as background in (Fig. biotinylated in detected only in the on-bead and in the A of the other background proteins in the on-bead These to the streptavidin and detected by the sensitive MS via chemical cleavage a reduction in background proteins relative to on-bead (Fig. identified in are the of and the of cathepsin targets of identified from a A of identified proteins is in in a new we to the of probe targets labeled via bioorthogonal or live cells of with and to click chemistry with alkyne biotin tag in labeling in cells in the detection of protein target in cell tandem MS identified cathepsin and in vitro and in and identified only in and to the (Fig. which the of their and Although cathepsin identified only in the on-bead of the targets in the for chemical release and on-bead The important of the cleavable linker is by the reduction of background proteins by to (Fig. identified in are the of and the of cathepsin targets of identified from a A of identified proteins is in in a new identified in are the of and the of cathepsin targets of identified from with live A of identified proteins is in in a new in have to the study of small modifications of proteins including post-translational modifications and For the enrichment of such modified the system is widely elution conditions are harsh and from A variety of cleavable linkers have been developed to the release of protein targets. of these mild cleavage Although this has in interesting for target and the of linker from (15Verhelst S.H.L. Fonovic M. Bogyo M. A mild chemically cleavable linker system for functional proteomic applications.Angew. Chem. Int. Ed. Engl. 2007; 46: 1284-1286Crossref PubMed Scopus (122) Google Scholar) to chemical P.P. Florea B.I. Li N. M.D. J. van der Marel G.A. Overkleeft H.S. A cleavable linker based on the for activity-based protein Chem. Int. Ed. 2010; 49: PubMed Scopus Google Scholar, C.A. Elias J.E. Gygi S.P. for quantitative proteomics Res. 2007; 6: PubMed Scopus Google Scholar). Here, we the application of which can be on in from as a cleavable linker which to in one is also commercially as inexpensive as Cleavable is with solid phase synthesis. the of the linker is a in the probe and the probe can be easy-to-handle peptide chemistry that be in an background in the enrichment of protein targets by is an essential in chemical proteomics might lead to contamination only by biotinylated but also by other the of the diol cleavable linker in target identification, we have used for the family of one probe that is and one probe that can be in with tandem that the elution of probe targets to cleaner and an to in background proteins relative to on-bead digestion. For new and probes, a reduction in the of will the necessary for target validation and the of target An of a cleavable linker is the of or probe-modified The of such modified will that the proteins are targets. Moreover, the site of modification can the of probe the active site of in as these are to of their M. Verhelst S.H.L. Sorum M.T. Bogyo M. Proteomics evaluation of chemically cleavable activity-based probes.Mol. Cell. Proteomics. 2007; 6: 1761-1770Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). to the in live cathepsin targets of the is that cathepsin is cell D. of cysteine in and J. PubMed Scopus Google Scholar). The of activity of and might be by the conditions or to The for lysates and cells the of target in living as the of small molecules natural products, or might be the of bioorthogonal in with the and release strategy by cleavable we have that a vicinal diol cleavable linker can be easily and into chemical The elution of probe targets in a efficient, and the target the linker to a reduction in background proteins. of with tandem labeling and target discovery in live we that the strategy will be of for other chemical biology and proteomics for M. Fonovic for and D. for general with