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Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês

10.1074/mcp.m113.031310

1535-9484

Lindsay D. Rogers, Christopher M. Overall,

Mass Spectrometry Techniques and Applications

Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events. Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events. Proteolysis involves the breakdown of proteins into smaller polypeptides or amino acids through the hydrolysis of peptide bonds by a protease. This represents a remarkably significant, but often underappreciated, post-translational modification (PTM) 1The abbreviations used are:COFRADICcombined fractional diagonal chromatographyC-TAILSC-terminal amine-based isotope labeling of substratesiMetinitiator methionineiTRAQisobaric tags for relative and absolute quantitationMSmass spectrometryN-AcN-terminal acetylationNATN-acetyltransferasePAGEpolyacrylamide gel electrophoresisPTMpost-translational modificationTAILSterminal amine isotope labeling of substratesTIStranslation initiation sitesTPA12-O-tetradecanoylphorbol 13-acetateuORFupstream open reading frameWTwild-type. 1The abbreviations used are:COFRADICcombined fractional diagonal chromatographyC-TAILSC-terminal amine-based isotope labeling of substratesiMetinitiator methionineiTRAQisobaric tags for relative and absolute quantitationMSmass spectrometryN-AcN-terminal acetylationNATN-acetyltransferasePAGEpolyacrylamide gel electrophoresisPTMpost-translational modificationTAILSterminal amine isotope labeling of substratesTIStranslation initiation sitesTPA12-O-tetradecanoylphorbol 13-acetateuORFupstream open reading frameWTwild-type. in that is it irreversible yet also ubiquitous. Consequently, the functional sequence of a protein can very rarely be predicted from its transcript, as proteolysis products form new (neo-) N and C termini. These cleavage events, or proteolytic processing events, can result in activation, inactivation, completely altered protein function, and even excision of "neo-proteins" with growth factor activity from an extracellular matrix parent molecule, and they regulate a vast array of biological processes (1Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. London, Academic Press1998Google Scholar). These include DNA replication, cell cycle progression, cell proliferation, and cell death, as well as pathological processes such as inflammation, cancer, arthritis, and cardiovascular disease. For example, in protein synthesis and maturation, precise selective removal of the N-terminal methionine and the signal peptide is essential for correct protein maturation and secretion. In some proteins, scission of the chain forms a molecule with four termini when linked by disulfide bridges. Through the removal of signal, nuclear, and mitochondrial localization sequences and ectodomain shedding, proteases regulate protein localization, and in viral infection, via cleavage of pre- and pro-domains and polyprotein processing, inactive proteins are converted into their active form(s), are inactivated, or change receptor-binding affinity. Thus, proteolysis is involved in much more than the mere degradation and turnover of proteins, important though these processes are in homeostasis. combined fractional diagonal chromatography C-terminal amine-based isotope labeling of substrates initiator methionine isobaric tags for relative and absolute quantitation mass spectrometry N-terminal acetylation N-acetyltransferase polyacrylamide gel electrophoresis post-translational modification terminal amine isotope labeling of substrates translation initiation sites 12-O-tetradecanoylphorbol 13-acetate upstream open reading frame wild-type. combined fractional diagonal chromatography C-terminal amine-based isotope labeling of substrates initiator methionine isobaric tags for relative and absolute quantitation mass spectrometry N-terminal acetylation N-acetyltransferase polyacrylamide gel electrophoresis post-translational modification terminal amine isotope labeling of substrates translation initiation sites 12-O-tetradecanoylphorbol 13-acetate upstream open reading frame wild-type. Proteases exist in all orders of life and constitute one of the largest enzyme families in humans (2Puente X.S. Sanchez L.M. Overall C.M. Lopez-Otin C. Human and mouse proteases: a comparative genomic approach.Nature Rev. Genet. 2003; 4: 544-558Crossref PubMed Scopus (745) Google Scholar), and more than 30 drugs targeting these enzymes are currently approved for clinical use (3Turk B. Targeting proteases: successes, failures and future prospects.Nat. Rev. Drug Discov. 2006; 5: 785-799Crossref PubMed Scopus (1042) Google Scholar). However, in order to fully comprehend the cellular function(s) of a given protease, one must have knowledge of the proteins processed by that protease, as well as the functions of these substrates and specific processing events. This is currently far from the case, as half of all human proteases have no known substrates (4Lopez-Otin C. Overall C.M. Protease degradomics: a new challenge for proteomics.Nat. Rev. Mol. Cell. Biol. 2002; 3: 509-519Crossref PubMed Scopus (627) Google Scholar). Degradomics is the application of high-throughput approaches to study proteases, their substrates, and their inhibitors on a system-wide scale (4Lopez-Otin C. Overall C.M. Protease degradomics: a new challenge for proteomics.Nat. Rev. Mol. Cell. Biol. 2002; 3: 509-519Crossref PubMed Scopus (627) Google Scholar). More specifically, terminomics is the specific characterization of protein N and C termini and, as such, forms a subfield of degradomics. This review provides an overview of current proteomics-based methods for characterizing protease cleavage events and protein termini. The quantitation, analysis, and curation of proteomics data, as well as exciting new applications within these fields, are also considered. Several array- and library-based methods have been developed to identify protease active site specificities. These include substrate phage display (5Matthews D.J. Wells J.A. Substrate phage: selection of protease substrates by monovalent phage display.Science. 1993; 260: 1113-1117Crossref PubMed Scopus (317) Google Scholar) and bacterial substrate display (6Boulware K.T. Daugherty P.S. Protease specificity determination by using cellular libraries of peptide substrates (CLiPS).Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 7583-7588Crossref PubMed Scopus (100) Google Scholar), whereby bacteriophages or bacteria express a chimeric cell surface protein containing a peptide of random sequence and an affinity tag. Proteolysis enables selection based on the affinity tag, and cleavable sequences are determined via DNA sequencing. However, these approaches do not provide the exact cleavage site in the random sequence; for this, a second step is required. Similarly, peptide libraries and microarrays have been used. For microarrays, arrayed peptide libraries are incubated with a test protease and cleavage is detected via methods such as loss of fluorophore binding or the removal of a fluorescent quencher (7Salisbury C.M. Maly D.J. Ellman J.A. Peptide microarrays for the determination of protease substrate specificity.J. Am. Chem. Soc. 2002; 124: 14868-14870Crossref PubMed Scopus (238) Google Scholar, 8Rosse G. Kueng E. Page M.G. Schauer-Vukasinovic V. Giller T. Lahm H.W. Hunziker P. Schlatter D. Rapid identification of substrates for novel proteases using a combinatorial peptide library.J. Comb. Chem. 2000; 2: 461-466Crossref PubMed Scopus (54) Google Scholar, 9Gosalia D.N. Salisbury C.M. Maly D.J. Ellman J.A. Diamond S.L. Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays.Proteomics. 2005; 5: 1292-1298Crossref PubMed Scopus (97) Google Scholar, 10Winssinger N. Damoiseaux R. Tully D.C. Geierstanger B.H. Burdick K. Harris J.L. PNA-encoded protease substrate microarrays.Chem. Biol. 2004; 11: 1351-1360Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Petrassi H.M. Williams J.A. Li J. Tumanut C. Ek J. Nakai T. Masick B. Backes B.J. Harris J.L. A strategy to profile prime and non-prime proteolytic substrate specificity.Bioorg. Med. Chem. Lett. 2005; 15: 3162-3166Crossref PubMed Scopus (40) Google Scholar, 12Loch C.M. Cuccherini C.L. Leach C.A. Strickler J.E. Deubiquitylase, deSUMOylase, and deISGylase activity microarrays for assay of substrate preference and functional modifiers.Mol. Cell. Proteomics. 2011; 10 (M110.002402)Abstract Full Text Full Text PDF PubMed Google Scholar). Library-based approaches are similar except that peptide mixtures are typically sequenced via Edman degradation or mass spectrometry (MS). One example is mixture-based oriented peptide libraries, which was the first approach used successfully to sequence the prime-side residues of the cleavage site in a library (13Turk B.E. Huang L.L. Piro E.T. Cantley L.C. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries.Nat. Biotechnol. 2001; 19: 661-667Crossref PubMed Scopus (463) Google Scholar). The prime-side cleavage motif (sequence C-terminal to the cleavage site) is determined by proteolysis of a library of N-terminally acetylated dodecamers sequenced via Edman degradation. Subsequently, a second library containing this predetermined prime-side sequence, a random unblocked N terminus, and a C-terminal biotin tag is generated and a second incubation with the protease is performed. Undigested peptides and C-terminal fragments are removed by means of avidin capture, and a second round of Edman degradation determines nonprime-side specificity. In view of the multiple time-consuming steps involved in generating custom second libraries in this otherwise very successful approach, new approaches have been sought to rapidly determine the prime-side and nonprime-side sequences in combination. Proteomic identification of protease cleavage sites (PICS) is one such approach (14Schilling 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). PICS employs a diverse, biologically relevant, and database-searchable peptide library generated from a cellular proteome using trypsin or Glu-C (14Schilling 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, 15Schilling O. Huesgen P.F. Barre O. Auf dem Keller U. Overall C.M. Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry.Nat. Protoc. 2011; 6: 111-120Crossref PubMed Scopus (84) Google Scholar). Primary amines (N-terminal α-amines and lysine ε-amines) are blocked, and this forms the library. A test protease is added, and the new terminal α-amines generated by proteolysis are selectively biotinylated and affinity purified. Purified peptides are sequenced via liquid-chromatography tandem mass spectrometry (LC-MS/MS) to determine prime-side cleavage motifs, whereas sequences N-terminal to cleavage sites are extracted bioinformatically. This can be done because the peptide library is accessible to conventional proteomics bioinformatics, whereas randomized synthetic peptide libraries are not. Thus, PICS enables the determination of both prime and nonprime cleavage site residues in the same experiment and so has the advantage of being very rapid. These peptide library-based techniques have been used to elucidate the cleavage site specificity of many proteases from all catalytic classes. However, a significant limitation is that they depend solely on amino acid sequences of relatively short peptides. Contributions of exosites and protein folding to cleavage site specificity cannot be observed, and as for all techniques that determine only the active site specificity, relevant in vivo protease substrates cannot be reliably identified solely from a cleavage site. Several proteomics methods have been developed to identify protease substrates. These include both two-dimensional polyacrylamide gel electrophoresis (PAGE)- and LC-MS/MS-based techniques. Following two-dimensional PAGE, stained spots are excised and identified via MS. Substrates are identified by a reduction in spot intensity of the intact protein and the appearance of spots corresponding to cleavage products (16Hwang I.K. Park S.M. Kim S.Y. Lee S.T. A proteomic approach to identify substrates of matrix metalloproteinase-14 in human plasma.Biochim. Biophys. Acta. 2004; 1702: 79-87Crossref PubMed Scopus (80) Google Scholar, 17Lee A.Y. Park B.C. Jang M. Cho S. Lee D.H. Lee S.C. Myung P.K. Park S.G. Identification of caspase-3 degradome by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight analysis.Proteomics. 2004; 4: 3429-3436Crossref PubMed Scopus (42) Google Scholar, 18Bredemeyer A.J. Lewis R.M. Malone J.P. Davis A.E. Gross J. Townsend R.R. Ley T.J. A proteomic approach for the discovery of protease substrates.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 11785-11790Crossref PubMed Scopus (112) Google Scholar). However, two-dimensional PAGE is restricted in terms of reproducibility and sensitivity, and it cannot be applied to small cleavage fragments or those differing by only a few residues. LC-MS/MS now provides vast improvements in throughput and proteome coverage. Shotgun proteomics has been used to identify substrates, including those whose localization has been altered by membrane shedding (19Tam E.M. Morrison C.J. Wu Y.I. Stack M.S. Overall C.M. Membrane protease proteomics: isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6917-6922Crossref PubMed Scopus (249) Google Scholar, 20Dean R.A. Butler G.S. Hamma-Kourbali Y. Delbe J. Brigstock D.R. Courty J. Overall C.M. Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.Mol. Cell. Biol. 2007; 27: 8454-8465Crossref PubMed Scopus (176) Google Scholar, 21Dean R.A. Overall C.M. Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome.Mol. Cell. Proteomics. 2007; 6: 611-623Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 22Butler G.S. Dean R.A. Tam E.M. Overall C.M. Pharmacoproteomics of a metalloproteinase hydroxamate inhibitor in breast cancer cells: dynamics of membrane type 1 matrix metalloproteinase-mediated membrane protein shedding.Mol. Cell. Biol. 2008; 28: 4896-4914Crossref PubMed Scopus (138) Google Scholar). This is done by comparing the secretomes of protease-treated cells to those of control cells. However, this approach cannot be used to determine the actual cleavage site. Nonetheless, these early labeling approaches utilizing isotope-coded affinity tags and isobaric tags for relative and absolute quantitation (iTRAQ) were very successful in easily identifying hundreds of biologically relevant substrates in the cellular context. Whereas these approaches are designed to determine substrates from complex proteomes, amino-terminal-oriented mass spectrometry of substrates is designed to identify multiple cleavage sites in proteins in vitro (23Doucet A. Overall C.M. Broad coverage identification of multiple proteolytic cleavage site sequences in complex high molecular weight proteins using quantitative proteomics as a complement to Edman sequencing.Mol. Cell. Proteomics. 2011; 10 (M110.003533)Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 24Doucet A. Overall C.M. Amino-terminal oriented mass spectrometry of substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry.Methods Enzymol. 2011; 501: 275-293Crossref PubMed Scopus (13) Google Scholar). Amino-terminal-oriented MS involves incubation of a purified substrate with a protease followed by dimethylation of the original and neo-N termini at the whole protein level. Subsequent trypsin digestion generates dimethylated semi-tryptic peptides containing the original N and C termini, as well as neo-N-terminal peptides representing cleavage sites that are identified by the dimethylated termini and their position in the protein sequence. The dimethylated cleavage sites are readily distinguished from the tryptic peptides, which contain a free primary amine at their N terminus. The protein topography and migration analysis platform uses one-dimensional SDS-PAGE in combination with LC-MS/MS to identify cleavage events by peptide mapping (25Dix M.M. Simon G.M. Cravatt B.F. Global mapping of the topography and magnitude of proteolytic events in apoptosis.Cell. 2008; 134: 679-691Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Here, proteins from protease-treated and control samples are resolved via one-dimensional SDS-PAGE, and each lane is cut into a number of gel slices, trypsinized, and analyzed via LC-MS/MS. Peptographs representing protein sequence coverage versus SDS migration identify proteolysis products based on shifts from higher to lower molecular weight species. This represents a development of an earlier study using gel slice analysis of isotopically labeled samples separated on one-dimensional SDS-PAGE (26Guo L. Eisenman J.R. Mahimkar R.M. Peschon J.J. Paxton R.J. Black R.A. Johnson R.S. A proteomic approach for the identification of cell-surface proteins shed by metalloproteases.Mol. Cell. Proteomics. 2002; 1: 30-36Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), but with the advantage of employing visually useful software for analysis. Like most gel-based approaches, this is very mass spectrometry intensive, and only occasionally is the exact cleavage site also directly identified. Recently, secretome protein enrichment with click sugars was developed (27Kuhn P.H. Koroniak K. Hogl S. Colombo A. Zeitschel U. Willem M. Volbracht C. Schepers U. Imhof A. Hoffmeister A. Haass C. Rossner S. Brase S. Lichtenthaler S.F. Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons.EMBO J. 2012; 31: 3157-3168Crossref PubMed Scopus (255) Google Scholar). This approach involves the metabolic labeling of N- and O-linked glycans, followed by a click reaction resulting in their biotinylation. Secreted proteins and shed extracellular membrane proteins are purified from contaminating serum proteins by means of avidin capture followed by in-gel digestion and LC-MS/MS. Secretome protein enrichment is particularly useful for cell culture experiments in which the cells have fastidious growth requirements and require serum. The serum glycoproteins are not metabolically labeled, and this enables simplification of the proteomic sample before analysis by separating the cell-derived metabolically labeled proteins from the glycoproteins in serum. However, only glycoprotein substrates can be identified. The above techniques have been useful in substrate identification. However, as the vast majority of identified peptides are internal, except with the amino-terminal-oriented MS procedure, precise cleavage sites are rarely determined, especially for proteins identified with low sequence coverage. N-terminal PTMs, including proteolytic processing, can greatly influence the localization and activity of many proteins (28Lange P.F. Overall C.M. Protein TAILS: when termini tell tales of proteolysis and function.Curr. Opin. Chem. Biol. 2013; 17: 73-82Crossref PubMed Scopus (74) Google Scholar). For example, N-terminal acetylation (N-Ac) plays important roles in protein function, localization, and stability (29Hollebeke J. Van Damme P. Gevaert K. N-terminal acetylation and other functions of Nalpha-acetyltransferases.Biol. Chem. 2012; 393: 291-298Crossref PubMed Scopus (32) Google Scholar), and N-terminal methylation regulates protein–protein interactions (30Chen T. Muratore T.L. Schaner-Tooley C.E. Shabanowitz J. Hunt D.F. Macara I.G. N-terminal alpha-methylation of RCC1 is necessary for stable chromatin association and normal mitosis.Nat. Cell Biol. 2007; 9: 596-603Crossref PubMed Scopus (108) Google Scholar). Thus, characterizing protein N termini not only identifies protease cleavage sites, but also is important in determining the functional physiochemical properties of a proteome. Methods employing both positive and negative selection of N-terminal peptides were developed (Table I) following the early recognition that "keeping it simple" approaches aiming to identify rare semi-tryptic terminal peptides within a complex mixture of tryptic peptides without enrichment will not lead to proteome-wide coverage and so will miss most cleavage sites. This is especially relevant for low-abundance but biologically interesting proteins such as cytokines. Thus, a variety of terminal peptide enrichment strategies have been developed to improve both the coverage and the dynamic range of terminal peptide identifications.Table IMethods for enriching protein N and C terminiMethodaPublished name or description of enrichment method.Advantages/disadvantagesQuantitationReference(s)Selective enzymatic biotinylation of N termini(+) Positive selection of unmodified N terminiiTRAQ, SILAC, label-free selected reaction monitoring(32Mahrus S. Trinidad J.C. Barkan D.T. Sali A. Burlingame A.L. Wells J.A. Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini.Cell. 2008; 134: 866-876Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 33Yoshihara H.A. Mahrus S. Wells J.A. Tags for labeling protein N-termini with subtiligase for proteomics.Bioorg. Med. Chem. Lett. 2008; 18: 6000-6003Crossref PubMed Scopus (42) Google Scholar, 61Agard N.J. Mahrus S. Trinidad J.C. Lynn A. Burlingame A.L. Wells J.A. Global kinetic analysis of proteolysis via quantitative targeted proteomics.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 1913-1918Crossref PubMed Scopus (91) Google Scholar, 86Wildes D. Wells J.A. Sampling the N-terminal proteome of human blood.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 4561-4566Crossref PubMed Scopus (97) Google Scholar, 87Agard N.J. Maltby D. Wells J.A. Inflammatory stimuli regulate caspase substrate profiles.Mol. Cell. Proteomics. 2010; 9: 880-893Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar)(+) Does not require chemical modification(−) Requires expensive patent protected enzyme(−) Requires large amounts of sampleN-CLAP(+) Positive selection of unmodified N terminiNone to date(34Xu G. Shin S.B. Jaffrey S.R. Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 19310-19315Crossref PubMed Scopus (79) Google Scholar)(−) Enriched peptides are shortened by one residue(−) Not compatible with chemical stable-isotope labelingCOFRADIC(+) Negative selection of N and C termini12C4 and 13C4 butyric acid, NHS-13C2D3, SILAC, trypsin-catalyzed 18O exchange(35Gevaert K. Goethals M. Martens L. Van Damme J. Staes A. Thomas G.R. Vandekerckhove J. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.Nat. Biotechnol. 2003; 21: 566-569Crossref PubMed Scopus (504) Google Scholar, 36Staes A. Van Damme P. Helsens K. Demol H. Vandekerckhove J. Gevaert K. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).Proteomics. 2008; 8: 1362-1370Crossref PubMed Scopus (129) Google Scholar, 55Van 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, 88Van Damme P. Van Damme J. Demol H. Staes A. Vandekerckhove J. Gevaert K. A review of COFRADIC techniques targeting protein N-terminal acetylation.BMC Proc. 2009; 3 (Suppl 6): S6Crossref PubMed Google Scholar, 89Van Damme P. Martens L. Van Damme J. Hugelier K. Staes A. Vandekerckhove J. Gevaert K. Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis.Nat. Methods. 2005; 2: 771-777Crossref PubMed Scopus (205) Google Scholar, 90Gevaert K. Van Damme P. Ghesquiere B. Vandekerckhove J. Protein processing and other modifications analyzed by diagonal peptide chromatography.Biochim. Biophys. Acta. 2006; 1764: 1801-1810Crossref PubMed Scopus (32) Google Scholar)(−) Extensive fractionation enhances sample loss(−) >50 fractions/sample, making it very instrument intensive(−) Loss of His- and Arg-containing peptides during strong cation exchange chromatographyTAILS(+) Negative selection of modified and unmodified N-terminiStable-isotope dimethyl labeling, iTRAQ(37Kleifeld O. Doucet A. auf dem Keller U. Prudova A. Schilling O. Kainthan R.K. Starr A.E. Foster L.J. Kizhakkedathu J.N. Overall C.M. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.Nat. Biotechnol. 2010; 28: 281-288Crossref PubMed Scopus (405) Google Scholar, 79auf dem Keller U. Prudova A. Eckhard U. Fingleton B. Overall C.M. Systems-level analysis of proteolytic events in increased vascular permeability and complement activation in skin inflammation.Sci. Signal. 2013; 6: rs2Crossref PubMed Scopus (96) Google Scholar, 80Tholen S. Biniossek M.L. Gansz M. Gomez-Auli A. Bengsch F. Noel A. Kizhakkedathu J.N. Boerries M. Busch H. Reinheckel T. Schilling O. Deletion of cysteine cathepsins B or L yields differential impacts on murine skin proteome and degradome.Mol. Cell. Proteomics. 2013; 12: 611-625Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 91Prudova A. auf dem Keller U. Butler G.S. Overall C.M. Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.Mol. Cell. Proteomics. 2010; 9: 894-911Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 92Wilson C.H. Indarto D. Doucet A. Pogson L.D. Pitman M.R. Menz R.I. McNicholas K. Overall C.M. Abbott C.A. Identifying natural substrates for dipeptidyl peptidase 8 (DP8) and DP9 using terminal amine isotopic labelling of substrates, TAILS, reveals in vivo roles in cellular homeostasis and energy metabolism.J. Biol. Chem. 2013; 288: 13936-13949Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar)(+) Very low nonspecific binding to polymer(−) Requires commercially available hyperbranched polyglycerol aldehyde polymerPTAG(+) Negative selection of modified and unmodified N terminiNone to date(40Mommen G.P. van de Waterbeemd B. Meiring H.D. Kersten G. Heck A.J. de Jong A.P. Unbiased selective isolation of protein N-terminal peptides from complex proteome samples using phospho tagging (PTAG) and TiO(2)-based depletion.Mol. Cell. Proteomics. 2012; 11: 832-842Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar)(−) Loss of phosphorylated N-terminal peptides(−) Losses due to nonspecific binding to TiO2 materialEnrichment of modified N termini by selective α-amine biotinylation(+) Negative selection of modified N terminiNone to date(41Zhang X. Ye J. Hojrup P. A proteomics approach to study in vivo protein N(alpha)-modifications.J. Proteomics. 2009; 73: 240-251Crossref PubMed Scopus (27) Google Scholar, 42Zhang X. Ye J. Engholm-Keller K. Hojrup P. A proteome-scale study on in vivo protein Nalpha-acetylation using an optimized method.Proteomics. 2011; 11: 81-93Crossref PubMed Scopus (31) Google Scholar)(−) No retention of unmodified N termini(−) Loss of His-containing peptidesC-TAILS(+) Negative selection of modified and unmodified C terminiStable-isotope dimethyl labeling(54Schilling O. Barre 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)(+) Chemical tag identifies unmodified C termini(−) Difficult to achieve complete labeling of carboxyl groupsSILAC, stable isotope labeling by amino acids in cell culture.a Published name or description of enrichment method. Open table in a new tab SILAC, stable isotope labeling by amino