Proteomics Evaluation of Chemically Cleavable Activity-based Probes

Abstract

Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system. Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system. Over the last decade, activity-based proteomics has become a prominent method for profiling enzyme activities in complex protein samples (for reviews, see Refs. 1Berger A.B. Vitorino P.M. Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery.Am. J. Pharmacogenomics. 2004; 4: 371-381Crossref PubMed Scopus (101) Google Scholar, 2Evans M.J. Cravatt B.F. Mechanism-based profiling of enzyme families.Chem. Rev. 2006; 106: 3279-3301Crossref PubMed Scopus (458) Google Scholar, 3Fonović M. Bogyo M. Activity based probes for proteases: applications to biomarker discovery, molecular imaging and drug screening.Curr. Pharm. Des. 2007; 13: 253-261Crossref PubMed Scopus (109) Google Scholar, 4Jeffery D.A. Bogyo M. Chemical proteomics and its application to drug discovery.Curr. Opin. Biotechnol. 2003; 14: 87-95Crossref PubMed Scopus (204) Google Scholar, 5Sadaghiani A.M. Verhelst S.H. Bogyo M. Tagging and detection strategies for activity-based proteomics.Curr. Opin. Chem. Biol. 2007; 11: 20-28Crossref PubMed Scopus (185) Google Scholar). This approach is dependent on small molecules termed activity-based probes (ABPs) 1The abbreviations used are: ABP, activity-based probe; DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMF, N,N-dimethylformamide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1. 1The abbreviations used are: ABP, activity-based probe; DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMF, N,N-dimethylformamide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1. that use chemically reactive functional groups to covalently modify the active sites of a specific enzyme class. Activity-based probes have been specifically designed to target enzyme families with well established catalytic mechanisms including proteases, kinases, lipases, glycosylases, histone deacylases, and phosphatases (for a general review, see Ref. 2Evans M.J. Cravatt B.F. Mechanism-based profiling of enzyme families.Chem. Rev. 2006; 106: 3279-3301Crossref PubMed Scopus (458) Google Scholar). Among proteases, activity-based probes have been mainly used for profiling the serine and cysteine families because both utilize catalytic nucleophiles in their active site that can be effectively targeted by a number of classes of synthetically accessible electrophiles (6Bogyo M. Verhelst S. Bellingard-Dubouchaud V. Toba S. Greenbaum D. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs.Chem. Biol. 2000; 7: 27-38Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 8Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Epoxide electrophiles cysteine profiling and Biol. 2000; 7: Full Text Full Text PDF PubMed Scopus Google Scholar, D. A.B. T. Bogyo M. Activity-based probes that target cysteine Chem. Biol. PubMed Scopus Google Scholar, Cravatt B.F. Activity-based protein profiling: the serine S. A. PubMed Scopus Google Scholar, S. Selective functional probes of and is a of of target Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D.A. J. Bogyo M. A. of activity-based probes for serine Chem. 2006; PubMed Scopus Google of ABPs is that of the of enzyme that can be used to the of targets. ABPs efficient to complex is used a to isolation of proteins by affinity on is used because is cheap and to proteins to be with of affinity with is the conditions to the these conditions are compatible with mass samples be by and proteins be from a for This to significant of and can for proteins that well proteins can be from the by and proteins can be significant and in detection of targets. the on-bead digestion method to a of of proteins to the These are problematic because is the and of proteomics of the of cleavable have been These M. of a novel for the selective of and PubMed Scopus Google that can be by a enzyme Cravatt B.F. for Chem. PubMed Scopus Google and M. J. T. chemically cleavable in the of from affinity S. A. PubMed Scopus Google Scholar). of these have for specific have that their general elution by is and be from the to analysis. elution of and to be because elution is dependent on that utilize a linker are compatible with reagents and are under have a linker that can be to the using a mild S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). the of cleavable linker to ABPs that target both cysteine and serine proteases. with the on-bead digestion of specific elution proteomics data that are of protein have the of the that specifically targets cysteine proteases S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). This has and a biotin and the cleavable linker that can be specifically with elution using mild conditions S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). cleavable ABP, target cysteine proteases are using and by of the linker using to use general a to the of the cleavable linker for activity-based protein profiling using standard activity-based probes with proteins be for in of can be by by in the of by and can be on digestion is and is to digestion is of the used for proteomics of targets J. of and a for Biol. 2006; PubMed Scopus Google Scholar, N. S. M. M. Cravatt B.F. for functional proteomics of PubMed Scopus Google Scholar, M. for of in samples with a of affinity and mass Chem. 2003; PubMed Scopus Google Scholar, M. affinity for purification under application in ubiquitin profiling and protein complex with in vivo 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are and are to affinity However, and proteins can be the digestion after These proteins and can with of of the significant of the cleavable linker is that from proteins. However, for the cleavable linker to be of efficient elution of targets. the of the cleavable we samples of proteins by on-bead digestion and by and with the and One on the proteins chemically from the by linker and in under a small of with the related This allows of by by of the with a the in and the of proteins that by with the general to the for of and by the related D. A. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical for the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with these of and on the its molecular based on be both and H. This be due to in based on on-bead digestion samples with proteins. main is a proteins and in and of to proteins. in due to the of in obtained from on the a number of proteins of cysteine proteases D. A. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical for the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the targets for both that elution is for S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). a of the of the linker on-bead digestion of the to of the target proteins of with these results that of the linker is efficient and results in significant in by of target by on-bead digestion and of the cleavable that allows of probe-labeled proteins by by with a and with for and by the for mass spectrometric protein that are by with the cysteine on of the One for by on-bead the chemically and in in are with their number and molecular compare both the number of and of are for that molecular of in the are for and are the of active on the the cleavable to to the and in and samples be in that have in activity-based protein profiling of with the of cysteine activities that be by on their molecular and these proteases to be and on-bead digestion of a of cysteine proteases and ubiquitin carboxyl-terminal hydrolase of the chemically and of these cysteine proteases and and ubiquitin carboxyl-terminal hydrolase are members of the of cysteine proteases, targets of the However, is a protein that of protein K.D. S. J. protein is a ubiquitin carboxyl-terminal PubMed Scopus Google a the of and the to UCH-L1, we that is in a protein that in the on-bead the protein that can be by in the in the on-bead digestion the method both of a protein that to be a target and to a target that by on-bead digestion due to that of probe-labeled that the the of quality in the of targets in with for and with for mass spectrometric by affinity to and by on proteins with and on-bead digestion are with their number of and for both in the by elution are with their and and of the cleavable linker approach for profiling of catalytic classes of proteases in the of we the linker the of the to the is a that has been to target the serine proteases and in J. of serine proteases by of PubMed Scopus Google Scholar). However, has been used for proteomics used to and the by both and by on-bead digestion the on-bead a number of and the of the serine the of by elution used for the the number of with the on-bead we a with of has been on a functional and has been to be active the 2006; PubMed Scopus Google Scholar). is we to the of targets in the on-bead digestion that the be selective for targets that are active in of the cleavable linker for of serine in of the that the cleavable linker and is in to the serine with and by by biotin of proteins by of on chemically proteins from with protein the target after cleavage. number of proteins in the on proteins from the in the in the in based on molecular are using to the number of the in the in we to see the active site that are with the be and by analysis. This is for applications of the cleavable linker to probe-labeled are are the active site of of the cysteine are to due to their we the on on-bead digestion of active site by and active site from the in molecular mass of the and its the mass of to the active site cysteine to the These data that the active site cysteine and that be used to specifically and the active site in the target proteases. of and we from the of the of proteases These data with the finding that the of proteins in the of the the samples by and that active of active site of and S. with and to on by to site active site to the and specifically by with the of active site of both and that the with S. to the molecular mass of the active site of and are in the of the and are of of in and chemically are the of target are in of the to the of of the targets that of the proteases by the results using multiple activity-based probes that the chemically cleavable linker that these probes be for proteomics profiling of active serine and cysteine proteases. of cleavable linker is its to in in the number of both and linker have for of small proteomics probes that on affinity purification to the and of proteomics the presented we the of significant of the of targets we to with general probes are used and targets are these chemically cleavable be Over the last decade, activity-based proteomics has become a prominent method for profiling enzyme activities in complex protein samples (for reviews, see Refs. 1Berger A.B. Vitorino P.M. Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery.Am. J. Pharmacogenomics. 2004; 4: 371-381Crossref PubMed Scopus (101) Google Scholar, 2Evans M.J. Cravatt B.F. Mechanism-based profiling of enzyme families.Chem. Rev. 2006; 106: 3279-3301Crossref PubMed Scopus (458) Google Scholar, 3Fonović M. Bogyo M. Activity based probes for proteases: applications to biomarker discovery, molecular imaging and drug screening.Curr. Pharm. Des. 2007; 13: 253-261Crossref PubMed Scopus (109) Google Scholar, 4Jeffery D.A. Bogyo M. Chemical proteomics and its application to drug discovery.Curr. Opin. Biotechnol. 2003; 14: 87-95Crossref PubMed Scopus (204) Google Scholar, 5Sadaghiani A.M. Verhelst S.H. Bogyo M. Tagging and detection strategies for activity-based proteomics.Curr. Opin. Chem. Biol. 2007; 11: 20-28Crossref PubMed Scopus (185) Google Scholar). This approach is dependent on small molecules termed activity-based probes (ABPs) 1The abbreviations used are: ABP, activity-based probe; DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMF, N,N-dimethylformamide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1. 1The abbreviations used are: ABP, activity-based probe; DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMF, N,N-dimethylformamide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1. that use chemically reactive functional groups to covalently modify the active sites of a specific enzyme class. Activity-based probes have been specifically designed to target enzyme families with well established catalytic mechanisms including proteases, kinases, lipases, glycosylases, histone deacylases, and phosphatases (for a general review, see Ref. 2Evans M.J. Cravatt B.F. Mechanism-based profiling of enzyme families.Chem. Rev. 2006; 106: 3279-3301Crossref PubMed Scopus (458) Google Scholar). Among proteases, activity-based probes have been mainly used for profiling the serine and cysteine families because both utilize catalytic nucleophiles in their active site that can be effectively targeted by a number of classes of synthetically accessible electrophiles (6Bogyo M. Verhelst S. Bellingard-Dubouchaud V. Toba S. Greenbaum D. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs.Chem. Biol. 2000; 7: 27-38Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 8Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Epoxide electrophiles cysteine profiling and Biol. 2000; 7: Full Text Full Text PDF PubMed Scopus Google Scholar, D. A.B. T. Bogyo M. Activity-based probes that target cysteine Chem. Biol. PubMed Scopus Google Scholar, Cravatt B.F. Activity-based protein profiling: the serine S. A. PubMed Scopus Google Scholar, S. Selective functional probes of and is a of of target Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D.A. J. Bogyo M. A. of activity-based probes for serine Chem. 2006; PubMed Scopus Google Scholar). One of ABPs is that of the of enzyme that can be used to the of targets. ABPs efficient to complex is used a to isolation of proteins by affinity on is used because is cheap and to proteins to be with of affinity with is the conditions to the these conditions are compatible with mass samples be by and proteins be from a for This to significant of and can for proteins that well proteins can be from the by and proteins can be significant and in detection of targets. the on-bead digestion method to a of of proteins to the These are problematic because is the and of proteomics of the of cleavable have been These M. of a novel for the selective of and PubMed Scopus Google that can be by a enzyme Cravatt B.F. for Chem. PubMed Scopus Google and M. J. T. chemically cleavable in the of from affinity S. A. PubMed Scopus Google Scholar). of these have for specific have that their general elution by is and be from the to analysis. elution of and to be because elution is dependent on that utilize a linker are compatible with reagents and are under have a linker that can be to the using a mild S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). the of cleavable linker to ABPs that target both cysteine and serine proteases. with the on-bead digestion of specific elution proteomics data that are of protein have the of the that specifically targets cysteine proteases S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). This has and a biotin and the cleavable linker that can be specifically with elution using mild conditions S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). cleavable ABP, target cysteine proteases are using and by of the linker using to use general a to the of the cleavable linker for activity-based protein profiling using standard activity-based probes with proteins be for in of can be by by in the of by and can be on digestion is and is to digestion is of the used for proteomics of targets J. of and a for Biol. 2006; PubMed Scopus Google Scholar, N. S. M. M. Cravatt B.F. for functional proteomics of PubMed Scopus Google Scholar, M. for of in samples with a of affinity and mass Chem. 2003; PubMed Scopus Google Scholar, M. affinity for purification under application in ubiquitin profiling and protein complex with in vivo 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are and are to affinity However, and proteins can be the digestion after These proteins and can with of of the significant of the cleavable linker is that from proteins. However, for the cleavable linker to be of efficient elution of targets. the of the cleavable we samples of proteins by on-bead digestion and by and with the and One on the proteins chemically from the by linker and in under a small of with the related This allows of by by of the with a the in and the of proteins that by with the general to the for of and by the related D. A. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical for the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with these of and on the its molecular based on be both and H. This be due to in based on on-bead digestion samples with proteins. main is a proteins and in and of to proteins. in due to the of in obtained from on the a number of proteins of cysteine proteases D. A. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical for the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the targets for both that elution is for S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). a of the of the linker on-bead digestion of the to of the target proteins of with these results that of the linker is efficient and results in significant in by the cleavable to to the and in and samples be in that have in activity-based protein profiling of with the of cysteine activities that be by on their molecular and these proteases to be and on-bead digestion of a of cysteine proteases and ubiquitin carboxyl-terminal hydrolase of the chemically and of these cysteine proteases and and ubiquitin carboxyl-terminal hydrolase are members of the of cysteine proteases, targets of the However, is a protein that of protein K.D. S. J. protein is a ubiquitin carboxyl-terminal PubMed Scopus Google a the of and the to UCH-L1, we that is in a protein that in the on-bead the protein that can be by in the in the on-bead digestion the method both of a protein that to be a target and to a target that by on-bead digestion due to that of probe-labeled that the the of quality in the of targets in with for and with for mass spectrometric by affinity to and by on proteins with and on-bead digestion are with their number of and for both in the by elution are with their and and of the cleavable linker approach for profiling of catalytic classes of proteases in the of we the linker the of the to the is a that has been to target the serine proteases and in J. of serine proteases by of PubMed Scopus Google Scholar). However, has been used for proteomics used to and the by both and by on-bead digestion the on-bead a number of and the of the serine the of by elution used for the the number of with the on-bead we a with of has been on a functional and has been to be active the 2006; PubMed Scopus Google Scholar). is we to the of targets in the on-bead digestion that the be selective for targets that are active in of the cleavable linker for of serine in of the that the cleavable linker and is in to the serine with and by by biotin of proteins by of on chemically proteins from with protein the target after cleavage. number of proteins in the on proteins from the in the in the in based on molecular are using to the number of the in the in we to see the active site that are with the be and by analysis. This is for applications of the cleavable linker to probe-labeled are are the active site of of the cysteine are to due to their we the on on-bead digestion of active site by and active site from the in molecular mass of the and its the mass of to the active site cysteine to the These data that the active site cysteine and that be used to specifically and the active site in the target proteases. of and we from the of the of proteases These data with the finding that the of proteins in the of the the samples by and that active of active site of and S. with and to on by to site active site to the and specifically by with the of active site of both and that the with S. to the molecular mass of the active site of and are in the of the and are of of in and chemically are the of target are in of the to the of of the targets that of the proteases by the results using multiple activity-based probes that the chemically cleavable linker that these probes be for proteomics profiling of active serine and cysteine proteases. of cleavable linker is its to in in the number of both and linker have for of small proteomics probes that on affinity purification to the and of proteomics the presented we the of significant of the of targets we to with general probes are used and targets are these chemically cleavable be have the of the that specifically targets cysteine proteases S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). This has and a biotin and the cleavable linker that can be specifically with elution using mild conditions S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). cleavable ABP, target cysteine proteases are using and by of the linker using to use general a to the of the cleavable linker for activity-based protein profiling using standard activity-based probes with proteins be for in of can be by by in the of by and can be on digestion is and is to digestion is of the used for proteomics of targets J. of and a for Biol. 2006; PubMed Scopus Google Scholar, N. S. M. M. Cravatt B.F. for functional proteomics of PubMed Scopus Google Scholar, M. for of in samples with a of affinity and mass Chem. 2003; PubMed Scopus Google Scholar, M. affinity for purification under application in ubiquitin profiling and protein complex with in vivo 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are and are to affinity However, and proteins can be the digestion after These proteins and can with of targets. One of the significant of the cleavable linker is that from proteins. However, for the cleavable linker to be of efficient elution of targets. the of the cleavable we samples of proteins by on-bead digestion and by and with the and One on the proteins chemically from the by linker and in under a small of with the related This allows of by by of the with a the in and the of proteins that by with the general to the for of and by the related D. A. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical for the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with these of and on the its molecular based on be both and H. This be due to in based on on-bead digestion samples with proteins. main is a proteins and in and of to proteins. in due to the of in obtained from on the a number of proteins of cysteine proteases D. A. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical for the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the targets for both that elution is for S.H. M. Bogyo M. mild chemically cleavable linker for functional Chem. 2007; PubMed Scopus Google Scholar). a of the of the linker on-bead digestion of the to of the target proteins of with these results that of the linker is efficient and results in significant in by proteins. the cleavable to to the and in and samples be in that have in activity-based protein profiling of with the of cysteine activities that be by on their molecular and these proteases to be and on-bead digestion of a of cysteine proteases and ubiquitin carboxyl-terminal hydrolase of the chemically and of these cysteine proteases and and ubiquitin carboxyl-terminal hydrolase are members of the of cysteine proteases, targets of the However, is a protein that of protein K.D. S. J. protein is a ubiquitin carboxyl-terminal PubMed Scopus Google a the of and the to UCH-L1, we that is in a protein that in the on-bead the protein that can be by in the in the on-bead digestion the method both of a protein that to be a target and to a target that by on-bead digestion due to that of probe-labeled that the the of quality in the of the cleavable linker approach for profiling of catalytic classes of proteases in the of we the linker the of the to the is a that has been to target the serine proteases and in J. of serine proteases by of PubMed Scopus Google Scholar). However, has been used for proteomics used to and the by both and by on-bead digestion the on-bead a number of and the of the serine the of by elution used for the the number of with the on-bead we a with of has been on a functional and has been to be active the 2006; PubMed Scopus Google Scholar). is we to the of targets in the on-bead digestion that the be selective for targets that are active in we to see the active site that are with the be and by analysis. This is for applications of the cleavable linker to probe-labeled are are the active site of of the cysteine are to due to their we the on on-bead digestion of active site by and active site from the in molecular mass of the and its the mass of to the active site cysteine to the These data that the active site cysteine and that be used to specifically and the active site in the target proteases. of and we from the of the of proteases These data with the finding that the of proteins in the of the the samples by and that active proteases. results using multiple activity-based probes that the chemically cleavable linker that these probes be for proteomics profiling of active serine and cysteine proteases. of cleavable linker is its to in in the number of both and linker have for of small proteomics probes that on affinity purification to the and of proteomics the presented we the of significant of the of targets we to with general probes are used and targets are these chemically cleavable be for and