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• W2123245786 abstract "Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine. Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine. Carboxypeptidases (CPs) 1The abbreviations used are:ACEAngiotensin-converting enzymeACE2Angiotensin-converting enzyme 2Ang-IAngiotensin IBMMCsBone marrow-derived mast cellsCOFRADICCOmbined FRActional DIagonal ChromatographyCPsCarboxypeptidasesCPAsA-like carboxypeptidaseshCPA1Human carboxypeptidase A1hCPA2Human carboxypeptidase A2mCPA3Mouse carboxypeptidase A3hCPA4Human carboxypeptidase A4MC-CPAMast cell carboxypeptidaseMCPsMetallocarboxypeptidasesmMCP-5Mouse mast cell proteinase-5MSP-MSMultiplex substrate profiling by mass spectrometryPRCPProlylcarboxypeptidaseSCPsSerine carboxypeptidases. 1The abbreviations used are:ACEAngiotensin-converting enzymeACE2Angiotensin-converting enzyme 2Ang-IAngiotensin IBMMCsBone marrow-derived mast cellsCOFRADICCOmbined FRActional DIagonal ChromatographyCPsCarboxypeptidasesCPAsA-like carboxypeptidaseshCPA1Human carboxypeptidase A1hCPA2Human carboxypeptidase A2mCPA3Mouse carboxypeptidase A3hCPA4Human carboxypeptidase A4MC-CPAMast cell carboxypeptidaseMCPsMetallocarboxypeptidasesmMCP-5Mouse mast cell proteinase-5MSP-MSMultiplex substrate profiling by mass spectrometryPRCPProlylcarboxypeptidaseSCPsSerine carboxypeptidases. catalyze the release of C-terminal amino acids from proteins and peptides (1Vendrell J. Avilés F.X. Carboxypeptidases.in: Turk V. Proteases: new perspectives. Birkhauser, Basel1999: 13-34Crossref Google Scholar, 2Arolas J.L. Vendrell J. Aviles F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (66) Google Scholar), and are grouped according to the chemical nature of their catalytic site. Accordingly, there are three types of carboxypeptidases: metallocarboxypeptidases (MCPs), serine carboxypeptidases (SCPs), and cysteine carboxypeptidases. CPs can also be classified based on their substrate specificity; CPs that prefer hydrophobic C-terminal amino acids (A-like MCPs or C-type SCPs), those that cleave C-terminal basic residues (B-like MCPs or D-type SCPs), those that recognize substrates with C-terminal aspartate or glutamate residues, and other CPs that display a broad substrate specificity (3Lyons P.J. Fricker L.D. Carboxypeptidase O is a glycosylphosphatidylinositol-anchored intestinal peptidase with acidic amino acid specificity.J. Biol. Chem. 2011; 286: 39023-39032Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 4Mortensen U.H. Olesen K. Breddam K. Carboxypeptidase C including carboxypeptidase Y.in: Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of proteolytic enzymes. Academic Press, London1999: 389-393Google Scholar). Angiotensin-converting enzyme Angiotensin-converting enzyme 2 Angiotensin I Bone marrow-derived mast cells COmbined FRActional DIagonal Chromatography Carboxypeptidases A-like carboxypeptidases Human carboxypeptidase A1 Human carboxypeptidase A2 Mouse carboxypeptidase A3 Human carboxypeptidase A4 Mast cell carboxypeptidase Metallocarboxypeptidases Mouse mast cell proteinase-5 Multiplex substrate profiling by mass spectrometry Prolylcarboxypeptidase Serine carboxypeptidases. Angiotensin-converting enzyme Angiotensin-converting enzyme 2 Angiotensin I Bone marrow-derived mast cells COmbined FRActional DIagonal Chromatography Carboxypeptidases A-like carboxypeptidases Human carboxypeptidase A1 Human carboxypeptidase A2 Mouse carboxypeptidase A3 Human carboxypeptidase A4 Mast cell carboxypeptidase Metallocarboxypeptidases Mouse mast cell proteinase-5 Multiplex substrate profiling by mass spectrometry Prolylcarboxypeptidase Serine carboxypeptidases. CPs were initially considered as degrading enzymes associated with protein catabolism. However, accumulating evidence demonstrates that some CPs are (more) selective and play key roles in controlling various biological processes (2Arolas J.L. Vendrell J. Aviles F.X. Fricker L.D. Metallocarboxypeptidases: emerging drug targets in biomedicine.Curr. Pharm. Des. 2007; 13: 349-366Crossref PubMed Scopus (66) Google Scholar, 5Rodriguez de la Vega M. Sevilla R.G. Hermoso A. Lorenzo J. Tanco S. Diez A. Fricker L.D. Bautista J.M. Avilés F.X. Nna1-like proteins are active metallocarboxypeptidases of a new and diverse M14 subfamily.FASEB J. 2007; 21: 851-865Crossref PubMed Scopus (85) Google Scholar). Angiotensin-converting enzyme 2 (ACE2), a MCP homolog of angiotensin-converting enzyme (ACE) that belongs to the M2 family of proteolytic enzymes according to the MEROPS classification, is a potent negative regulator of the renin-angiotensin system and plays a key role in maintaining blood pressure homeostasis. ACE2 cleaves off a C-terminal phenylalanine thereby converting angiotensin II to the heptapeptide angiotensin-(1–7), a peptide hormone that opposes the vasoconstrictor and proliferative actions of angiotensin II (6Kuba K. Imai Y. Ohto-Nakanishi T. Penninger J.M. Trilogy of ACE2: a peptidase in the renin-angiotensin system, a SARS receptor, and a partner for amino acid transporters.Pharmacol. Ther. 2010; 128: 119-128Crossref PubMed Scopus (318) Google Scholar). Cathepsin A, a lysosomal SCP, is also believed to function in blood pressure regulation, in this case through its action against vasoactive peptides like endothelin-1 or angiotensin I (7Pshezhetsky A.V. Hinek A. Serine carboxypeptidases in regulation of vasoconstriction and elastogenesis.Trends Cardiovasc. Med. 2009; 19: 11-17Crossref PubMed Scopus (22) Google Scholar). Human carboxypeptidase A4 (hCPA4), a MCP from the M14 family, presumably functions in neuropeptide processing and was linked to prostate cancer aggressiveness (8Tanco S. Zhang X. Morano C. Avilés F.X. Lorenzo J. Fricker L.D. Characterization of the substrate specificity of human carboxypeptidase A4 and implications for a role in extracellular peptide processing.J. Biol. Chem. 2010; 285: 18385-18396Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Besides their biological importance, CPs are also exploited in biotechnological and biomedical applications. Carboxypeptidase B (CPB) for instance, is a M14 MCP used for manufacturing recombinant human insulin. Recombinant preproinsulin is enzymatically processed in vitro by pancreatic trypsin and carboxypeptidase B to generate the active insulin form (9Son Y.J. Kim C.K. Kim Y.B. Kweon D.H. Park Y.C. Seo J.H. Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B.Biotechnol. Prog. 2009; 25: 1064-1070Crossref PubMed Scopus (16) Google Scholar). Further, carboxypeptidase digestion has been used for determining the C-terminal sequence of purified proteins or peptides. The most popular CPs being the SCPs C, P and Y (10Bergman T. Cederlund E. Jornvall H. Fowler E. C-terminal sequence analysis.Curr. Protoc. Protein Sci. 2003; (Chapter 11, Unit 11.8)Crossref PubMed Scopus (1) Google Scholar). In addition, the food industry uses different SCPs to process protein products to reduce their bitter taste (11Raksakulthai R. Haard N.F. Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus).J. Agric. Food Chem. 2001; 49: 5019-5030Crossref PubMed Scopus (20) Google Scholar, 12Arai S. Noguchi M. Kurosawa S. Kato H. Fujimaki M. APPLYING PROTEOLYTIC ENZYMES ON SOYBEAN. 6. Deodorization Effect of Aspergillopeptidase A and Debittering Effect of Aspergillus Acid Carboxypeptidase.J. Food Sci. 1970; 35: 392-395Crossref Scopus (57) Google Scholar, 13Umetsu H. Matsuoka H. Ichishima E. Debittering mechanism of bitter peptides from milk casein by wheat carboxypeptidase.J. Agricultural Food Chem. 1983; 31: 50-53Crossref Scopus (62) Google Scholar). Identifying a protease's specificity and its natural substrates provides key information to understanding the molecular role of proteases (14Barrios A.M. Craik C.S. Scanning the prime-site substrate specificity of proteolytic enzymes: a novel assay based on ligand-enhanced lanthanide ion fluorescence.Bioorg. Med. Chem. Lett. 2002; 12: 3619-3623Crossref PubMed Scopus (23) Google Scholar, 15Gosalia D.N. Salisbury C.M. Ellman J.A. Diamond S.L. High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays.Mol. Cell Proteomics. 2005; 4: 626-636Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Moreover, determination of a protease's specificity also provides a framework for the design of selective probes and potent and selective inhibitors (16Diamond S.L. Methods for mapping protease specificity.Curr. Opin. Chem. Biol. 2007; 11: 46-51Crossref PubMed Scopus (94) Google Scholar). Although several factors impact on substrate selection, a key factor is the complementarity of a protease binding site with specific substrate side-chains. Several approaches for determining protease substrate specificity based on peptide libraries have been developed, including substrate phage/bacterial display libraries, peptide microarrays, positional-scanning peptide libraries, mixture-based peptide libraries, and proteome-derived peptide libraries (17auf dem Keller U. Schilling O. Proteomic techniques and activity-based probes for the system-wide study of proteolysis.Biochimie. 2010; 92: 1705-1714Crossref PubMed Scopus (47) Google Scholar). The latter were more recently introduced by Schilling et al. (18Schilling O. Overall C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites.Nat. Biotechnol. 2008; 26: 685-694Crossref PubMed Scopus (316) Google Scholar) and make use of natural peptide libraries generated by proteolysis of a model proteome using a specific protease (e.g. trypsin, chymotrypsin). Such peptide libraries are subsequently digested by a protease of interest and the resulting neo-N-terminal products are enriched and identified following LC-MS/MS analyses. This technology allows profiling of the substrate specificity of endoproteases and aminopeptidases. However, viewing the fact that only C-terminal cleavage products are isolated by this method, it cannot be used to study CPs because their resulting primed site cleavage products are typically only a single amino acid and thus are not compatible for subsequent LC-MS/MS based identification. Currently, two different peptide-centric degradomic approaches (19van den Berg B.H. Tholey A. Mass spectrometry-based proteomics strategies for protease cleavage site identification.Proteomics. 2012; 12: 516-529Crossref PubMed Scopus (30) Google Scholar) are available for CP substrate profiling. Recently, a multiplex substrate profiling by mass spectrometry (MSP-MS) method, which applies mass spectrometry-based peptide sequencing to detect cleavage products in a mixture of synthetic peptides, was used to determine the substrate preferences of prolylcarboxypeptidase (PRCP) (20O'Donoghue A.J. Eroy-Reveles A.A. Knudsen G.M. Ingram J. Zhou M. Statnekov J.B. Greninger A.L. Hostetter D.R. Qu G. Maltby D.A. Anderson M.O. Derisi J.L. McKerrow J.H. Burlingame A.L. Craik C.S. Global identification of peptidase specificity by multiplex substrate profiling.Nat. Methods. 2012; 9: 1095-1100Crossref PubMed Scopus (121) Google Scholar). Further, peptidomic studies have made use of natural peptides isolates from cells and tissues as natural substrate pools to test cleavages by CPs (8Tanco S. Zhang X. Morano C. Avilés F.X. Lorenzo J. Fricker L.D. Characterization of the substrate specificity of human carboxypeptidase A4 and implications for a role in extracellular peptide processing.J. Biol. Chem. 2010; 285: 18385-18396Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Lyons P.J. Fricker L.D. Substrate specificity of human carboxypeptidase A6.J. Biol. Chem. 2010; 285: 38234-38242Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 22Lyons P.J. Fricker L.D. Peptidomic approaches to study proteolytic activity.Curr. Protoc. Protein Sci. 2011; (Chapter 18, Unit18.13)Crossref PubMed Scopus (14) Google Scholar). In this list of degradomic approaches, we can additionally consider the protein-centric positional proteomics approaches; C-terminal COFRADIC (23Van Damme P. Staes A. Bronsoms S. Helsens K. Colaert N. Timmerman E. Aviles F.X. Vandekerckhove J. Gevaert K. Complementary positional proteomics for screening substrates of endo- and exoproteases.Nat. Methods. 2010; 7: 512-515Crossref PubMed Scopus (99) Google Scholar) and C-TAILS (24Schilling O. Barré O. Huesgen P.F. Overall C.M. Proteome-wide analysis of protein carboxy termini: C terminomics.Nat. Methods. 2010; 7: 508-511Crossref PubMed Scopus (134) Google Scholar), capable of identifying in vivo CP proteolytic events, based on the identification of protein neo-C termini. We here exploited the COFRADIC technology (25Gevaert K. Impens F. Van Damme P. Ghesquière B. Hanoulle X. Vandekerckhove J. Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses.FEBS J. 2007; 274: 6277-6289Crossref PubMed Scopus (22) Google Scholar) and developed a proteome-derived carboxypeptidase peptide library assay that was used to determine the substrate specificity profile of 5 selected human carboxypeptidases: 4 enzymes belonging to the MCP family and PRCP, which is a SCP. Given that MCPs are the most studied and thus a highly relevant group of CPs, the human metallocarboxypeptidases A4 (hCPA4), A2 (hCPA2), and A1 (hCPA1) were used as model CPs. Two different peptide libraries, created using chymotrypsin or metalloendopeptidase Lys-N as peptide library generating proteases, were used to extensively profile the proteolytic substrate specificities of these MCPs. In addition, we profiled the substrate preferences for the yet uncharacterized mast cell carboxypeptidase (MC-CPA or mCPA3). Besides, using Lys-N proteome-derived peptide libraries and making use of shorter protease incubation times, information on sequential cleavages of these enzymes could be obtained. Finally, this assay was additionally applied to PRCP, a pharmaceutically relevant SCP that differs from MCPs in its enzymatic characteristics, further demonstrating the more universal applicability of our method. Human K-562 cells were obtained from the American Type Culture Collection (CCL-243, ATCC, Manassas, VA, USA), and cultured in GlutaMAX™ containing RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen). Cells were maintained at 37 °C in a 5% CO2 humidified incubator. The human carboxypeptidases A1 (hCPA1), A2 (hCPA2) and A4 (hCPA4) were obtained as recombinant proteins using the pPIC9 expression vector and the methylotrophic yeast Pichia pastoris as an expression host. Enzyme purifications were performed as described previously (26Pallares I. Bonet R. Garcia-Castellanos R. Ventura S. Aviles F.X. Vendrell J. Gomis-Ruth F.X. Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 3978-3983Crossref PubMed Scopus (82) Google Scholar, 27Pallares I. Fernandez D. Comellas-Bigler M. Fernandez-Recio J. Ventura S. Aviles F.X. Bode W. Vendrell J. Direct interaction between a human digestive protease and the mucoadhesive poly(acrylic acid).Acta Crystallogr. D. 2008; 64: 784-791Crossref Scopus (14) Google Scholar, 28Reverter D. Garcia-Saez I. Catasus L. Vendrell J. Coll M. Avilés F.X. Characterisation and preliminary X-ray diffraction analysis of human pancreatic procarboxypeptidase A2.FEBS Lett. 1997; 420: 7-10Crossref PubMed Scopus (15) Google Scholar). These enzymes were purified in their zymogen form and the active enzymes were obtained through tryptic activation (at a 1/50 (w/w) ratio) for 1 h at room temperature. The resulting mature and activated enzymes were subsequently purified by anion-exchange chromatography (TSK-DEAE 5PW) on a FPLC-Äkta system using a linear salt gradient (ranging from 0 to 30% of 0.4 m ammonium acetate in 20 mm Tris-HCl, pH 9.0). Eluted fractions were analyzed by SDS-PAGE, and the purest fractions containing the enzyme were pooled, desalted, and concentrated to 1 mg/ml by Amicon centrifugal filter devices (Ultra 0.5 ml 10 kDa MWCO columns (Millipore, Billerica, MA, USA)). Mouse CPA3 (mCPA3) was purified from mouse bone marrow-derived mast cells (BMMCs) (kindly provided by Dr Gunnar Pejler, Swedish University of Agricultural Sciences) using a two-step purification procedure. Mast cells were lysed in 10 mm Tris-HCl, pH 7.4, 4 m NaCl, 0.1% PEG 3350 supplemented with a Complete EDTA-free Protease Inhibitor Mixture Tablet (Roche Diagnostics) (buffer A) for 30 min at 4 °C. The lysate was subsequently centrifuged and the supernatant was diluted twenty-fold in 50 mm Tris-HCl, pH 7.4 and 100 mm NaCl, supplemented with a Complete EDTA-free Protease Inhibitor Mixture Tablet (buffer B). The diluted extract was loaded on a Heparin HyperD® M column (Pall Biosepra, Cergy-Saint-Christophe, France) equilibrated with buffer B. mCPA3 was eluted using 5 volumes of buffer A. The eluate was concentrated on Amicon centrifugal filters devices, and fractionated on a Superdex 75 column in 25 mm Tris-HCl, pH 7.4, 1 m NaCl and 0.025% PEG 3350. The purest eluted fractions (analyzed by SDS-PAGE) showing the highest activity toward N-(4-methoxyphenylazoformyl)-Phe-OH (Bachem, Bubendorf, Switzerland) were pooled and concentrated by Amicon centrifugal filters devices (Ultra 0.5 ml 10 kDa MWCO columns, Millipore). Proteome-derived peptide libraries were generated from human K-562 cell extracts. Cells were repeatedly (3×) washed in digestion buffer (50 mm NH4CO3, pH 7.9) and re-suspended in this buffer at 2 × 107 cells per ml. Then, these cell suspensions were subjected to three rounds of freeze-thaw lysis and the lysate was cleared by centrifugation for 10 min at 16,000 × g at 4 °C. To prepare the chymotryptic and metalloendopeptidase Lys-N proteome-derived peptide libraries, the lysates were respectively digested for 4 h at 37 °C using sequencing-grade chymotrypsin (Promega, Madison, WI, USA) at an enzyme/substrate ratio of 1/200 (w/w) or recombinant Lys-N (1/85, w/w) for two hours at 37 °C (U-Protein Express BV, Utrecht, The Netherlands). To stop proteolytic digestion, acetic acid was added to a 4% final concentration. For the chymotryptic peptide library and to prevent oxidation of methionines between the primary and secondary RP-HPLC runs, methionines were oxidized before the primary run. The methionine oxidation reaction proceeded in the injector compartment by transferring 20 μl of a freshly prepared aqueous 3% H2O2 solution to a vial containing 90 μl of the acidified peptide mixture (final concentration of 0.54% H2O2). This reaction proceeded for 30 min at 30 °C after which the sample was immediately injected onto the RP-HPLC column. No prior methionine oxidation was performed when the Lys-N peptide libraries were assayed (see “Results”). From these mixtures, 100 μl (equivalent to ∼350 μg of digested proteins) was injected onto a RP-column (Zorbax 300SB-C18 Narrow Bore, 2.1 mm internal diameter (I.D.) x 150 mm length, 5 μm particles; Agilent Technologies) for the first RP-HPLC run. Following 10 min isocratic pumping with solvent A (10 mm ammonium acetate in water/acetonitrile (98/2, v/v), pH 5.5), a gradient was started of 1% solvent B (10 mm ammonium acetate in water/acetonitrile (30/70, v/v), pH 5.5) increase per minute. The column was then run at 100% solvent B for 5 min, switched to 100% solvent A and re-equilibrated for 20 min with solvent A. The flow was kept constant at 80 μl/min using Agilent's 1100 series capillary pump with an 800 μl/min flow controller. Twenty fractions of 2 min intervals (from 20 to 60 min after sample injection) and 26 fractions (from 20 to 72 min after sample injection) were collected for the chymotryptic library and the Lys-N library respectively. Each peptide fraction was dried and re-dissolved in 20 μl of CP-supplemented assay buffer (50 mm Tris-HCl, pH 8.0 and 100 mm NaCl prepared with 90% H218O water (Cambridge Isotope Labs, Andover, MA, USA) containing 7.3 units of MCP per ml (which is approximately equivalent to a 10 nm CP concentration)). Note that one unit of MCP activity was defined as the amount of enzyme hydrolyzing 1 nmol of N-(4-methoxyphenylazoformyl)-l-phenylalanine substrate per min at 37 °C. For PRCP (BPS Biosciences, San Diego, CA, USA), a 5 nm final assay concentration was used. CP hydrolysis was allowed to proceed for 2 h at 37 °C and stopped by addition of 33 μl of 4% acetic acid in solvent A. All 20 (chymotryptic library) or 26 (Lys-N library) samples were reloaded on the same RP-column and separated using identical conditions. Per sample, twenty secondary “shifted” fractions of 1 min wide were collected in a time interval ranging from 21 to 1 min before the fraction collection interval (but eluting the earliest at 10 min (start of the gradient)) used for the primary fraction. Whenever the “nonshifted” fractions were additionally analyzed, six extra 1 min wide fractions were collected in a time interval ranging from 1 min before to 3 min after the original fraction collection interval. All peptide fractions were dried and, secondary fractions eluting 4 min apart were pooled by re-dissolving these in a final volume of 20 μl of 2 mm TCEP and 2% acetonitrile, similar to a pooling strategy described previously (29Staes A. Impens F. Van Damme P. Ruttens B. Goethals M. Demol H. Timmerman E. Vandekerckhove J. Gevaert K. Selecting protein N-terminal peptides by combined fractional diagonal chromatography.Nat. Protoc. 2011; 6: 1130-1141Crossref PubMed Scopus (139) Google Scholar). In total, 40 (when shifted fractions were analyzed alone) or 52 (in the case the extra “nonshifted” fractions were additionally analyzed) peptide fractions per setup were subjected to LC-MS/MS analysis. LC-MS/MS analysis was performed using an Ultimate 3000 RSLC nano LC-MS/MS system (Dionex, Amsterdam, The Netherlands) in-line connected to a LTQ Orbitrap Velos (Thermo Fisher, Bremen, Germany). 2 μl of the sample mixture was first loaded on a trapping column (made in-house, 100 μm I.D. × 20 mm length, 5 μm Reprosil-Pur Basic-C18-HD beads, Dr. Maisch, Ammerbuch-Entringen, Germany). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 μm I.D. × 150 mm length, 3 μm C18 Reprosil-Pur Basic-C18-HD beads). Peptides were loaded with solvent A' (0.1% trifluoroacetic acid in 2% acetonitrile) and were separated with a linear gradient from 98% of solvent A″ (0.1% formic acid in 2% acetonitrile) to 50% of solvent B' (0.1% formic acid in 80% acetonitrile) with a linear gradient of a 1.8% solvent B' increase per minute at a flow rate of 300 nl/min followed by a steep increase to 100% of solvent B′. The Orbitrap Velos mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the ten most abundant peaks in a MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The ten most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 20 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.2.0, Matrix Science, www.matrixscience.com/Distiller.html). Although generating these peak lists, grouping of spectra was allowed with maximum intermediate retention time of 30 s and maximum intermediate scan count of 5. Grouping was done with a 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal-to-noise limit was set at 2. The generated MS/MS peak lists were then searched with Mascot using the Mascot Daemon interface (version 2.3, Matrix Science). The Mascot search parameters were set as follows. Searches were performed in the Swiss-Prot database with taxonomy set to human (either the 2011_05, 2011_06 or 2013_01 UniProtKB/Swiss-Prot database release containing respectively 20,286, 20,312 and 20,307 human protein sequence entries were used). Single 18O modification of peptide C termini, acetylation of protein N termini and pyroglutamate formation of N-terminal glutamine were set as variable modifications. Methionine oxidation to methionine-sulfoxide was set as fixed modification for the chymotryptic library assays and as a variable modification for the Lys-N library assays. For the chymotryptic library the enzyme was set to “none” (because up to 3 missed cleavages could be detected), whereas for the Lys-N library a semi-Lys-N/P (semi Lys-N specificity with lysine-proline cleavage allowed) was set as enzyme allowing for one missed cleavage. The mass tolerance on the precursor ion was set to 10 ppm and on fragment ions to 0.5 Da. The peptide charge was set to 1+, 2+ or 3+ and the instrument setting was put on ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 95% confidence, were withheld. According to the method described by Käll et al. (30Kall L. Storey J.D. MacCoss M.J. Noble W.S. Assigning significance to peptides identified by tandem mass spectrometry using decoy databases.J. Proteome Res. 2008; 7: 29-34Crossref PubMed Scopus (443) Google Scholar), the false discovery rate was estimated and was found <2% at the spectrum level and <3% at the peptide level. Identified MS/MS spectra are available on-line in the PRoteomics IDEntifications database (PRIDE) (31Martens L. Hermjakob H. Jones P. Adamski M. Taylor C. States D. Gevaert K. Vandekerckhove J. Apweiler R. PRIDE: the proteomics identifications database.Proteomics. 2005; 5: 3537-3545Crossref PubMed Scopus (436) Google Scholar) under the project entitled “Proteome-derived peptide libraries to study the substrate specificity profiles of carboxypeptidases” under the accessions 26851–26859, 27075–27076 and 28756–28757. Primed side residues of the full-length peptidic substrates were inferred using an in-house java-based script named PeptideRetriever. This command-based tool maps the identified peptides onto a database of choice in a protein accession dependent manner, enabling extraction of" @default.
• W2123245786 created "2016-06-24" @default.
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• W2123245786 date "2013-08-01" @default.
• W2123245786 modified "2023-10-02" @default.
• W2123245786 title "Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases" @default.
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