Abstract
Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Brown, J.L. & Roberts, W.K. Evidence that approximately eighty per cent of the soluble proteins from Ehrlich ascites cells are Nalpha-acetylated. J. Biol. Chem. 251, 1009–1014 (1976).
Dormeyer, W., Mohammed, S., Breukelen, B., Krijgsveld, J. & Heck, A.J. Targeted analysis of protein termini. J. Proteome Res. 6, 4634–4645 (2007).
Doucet, A., Butler, G.S., Rodriguez, D., Prudova, A. & Overall, C.M. Metadegradomics: toward in vivo quantitative degradomics of proteolytic post-translational modifications of the cancer proteome. Mol. Cell. Proteomics 7, 1925–1951 (2008).
Gevaert, K. et al. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat. Biotechnol. 21, 566–569 (2003).
Kuhn, K. et al. Isolation of N-terminal protein sequence tags from cyanogen bromide cleaved proteins as a novel approach to investigate hydrophobic proteins. J. Proteome Res. 2, 598–609 (2003).
McDonald, L., Robertson, D.H., Hurst, J.L. & Beynon, R.J. Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides. Nat. Methods 2, 955–957 (2005).
McDonald, L. & Beynon, R.J. Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization. Nat. Protoc. 1, 1790–1798 (2006).
Ji, C., Guo, N. & Li, L. Differential dimethyl labeling of N-termini of peptides after guanidination for proteome analysis. J. Proteome Res. 4, 2099–2108 (2005).
Timmer, J.C. et al. Profiling constitutive proteolytic events in vivo. Biochem. J. 407, 41–48 (2007).
Mahrus, S. et al. Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134, 866–876 (2008).
Staes, A. et al. Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC). Proteomics 8, 1362–1370 (2008).
Gupta, N. & Pevzner, P.A. False discovery rates of protein identifications: a strike against the two-peptide rule. J. Proteome Res. 8, 4173–4181 (2009).
Hsu, J.L., Huang, S.Y., Chow, N.H. & Chen, S.H. Stable-isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75, 6843–6852 (2003).
Kainthan, R., Muliawan, E., Hatzikiriakos, S. & Brooks, D. Synthesis, characterization, and viscoelastic properties of high molecular weight hyperbranched polyglycerols. Macromolecules 39, 7708–7717 (2006).
Van Damme, P. et al. Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat. Methods 2, 771–777 (2005).
Keller, A., Eng, J., Zhang, N., Li, X.J. & Aebersold, R. A uniform proteomics MS/MS analysis platform utilizing open XML file formats. Mol. Syst. Biol. 1, 2005.0017 (2005).
Minn, A.J. et al. Genes that mediate breast cancer metastasis to lung. Nature 436, 518–524 (2005).
Dean, 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 6, 611–623 (2007).
Dean, R.A. et al. 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. 27, 8454–8465 (2007).
McQuibban, G.A. et al. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 289, 1202–1206 (2000).
Schilling, O. & Overall, C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites. Nat. Biotechnol. 26, 685–694 (2008).
Enoksson, M. et al. Identification of proteolytic cleavage sites by quantitative proteomics. J. Proteome Res. 6, 2850–2858 (2007).
Dix, M.M., Simon, G.M. & Cravatt, B.F. Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134, 679–691 (2008).
Butler, G.S. & Overall, C.M. Proteomic identification of multitasking proteins in unexpected locations complicates drug targeting. Nat. Rev. Drug Discov. 8, 935–948 (2009).
Butler, G.S., Dean, R.A., Tam, E.M. & Overall, C.M. Pharmacoproteomics of a metalloproteinase hydroxamate inhibitor in breast cancer cells: Dynamics of matrix metalloproteinase-14 (MT1-MMP) mediated membrane protein shedding. Mol. Cell. Biol. 28, 4896–4914 (2008).
Basset, P. et al. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348, 699–704 (1990).
Andarawewa, K.L. et al. Dual stromelysin-3 function during natural mouse mammary tumor virus-ras tumor progression. Cancer Res. 63, 5844–5849 (2003).
Pei, D., Majmudar, G. & Weiss, S.J. Hydrolytic inactivation of a breast carcinoma cell-derived serpin by human stromelysin-3. J. Biol. Chem. 269, 25849–25855 (1994).
Ahmad, A. et al. Stromelysin 3: an independent prognostic factor for relapse-free survival in node-positive breast cancer and demonstration of novel breast carcinoma cell expression. Am. J. Pathol. 152, 721–728 (1998).
Fu, Y. & Lee, A.S. Glucose regulated proteins in cancer progression, drug resistance and immunotherapy. Cancer Biol. Ther. 5, 741–744 (2006).
He, J. & Baum, L.G. Presentation of galectin-1 by extracellular matrix triggers T cell death. J. Biol. Chem. 279, 4705–4712 (2004).
Tateda, K. et al. Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: potential role of neutrophils as immunoregulatory cells. Infect. Immun. 69, 2017–2024 (2001).
Tester, A.M. et al. LPS responsiveness and neutrophil chemotaxis in vivo require PMN MMP-8 activity. PLoS One 2, e312 (2007).
Tao, W.A. et al. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat. Methods 2, 591–598 (2005).
Chang, T.K., Jackson, D.Y., Burnier, J.P. & Wells, J.A. Subtiligase: a tool for semisynthesis of proteins. Proc. Natl. Acad. Sci. USA 91, 12544–12548 (1994).
Simon, G.M., Dix, M.M. & Cravatt, B.F. Comparative assessment of large-scale proteomic studies of apoptotic proteolysis. ACS Chem. Biol. 4, 401–408 (2009).
LeDissez, C., Wong, P. & Brooks, D. Analysis of surface aldehyde functions on surfactant-free polystyrene/polyacrolein latex. Macromolecules 29, 953–959 (1996).
Leitner, A. & Lindner, W. Chemistry meets proteomics: the use of chemical tagging reactions for MS-based proteomics. Proteomics 6, 5418–5434 (2006).
Veh, R., Corfield, A., Sander, M. & Schauer, R. Neuraminic acid-specific modifications and tritium labeling of gangliosides. Biochim. Biophys. Acta 486, 145–160 (1976).
Gasteiger, E. et al. Protein identification and analysis tools on the ExPASy server. in The Proteomics Protocols Handbook (ed. Walker, J.M.) 571–607 (Humana Press, Totowa, New Jersey, USA, 2005).
Butler, G.S., Tam, E.M. & Overall, C.M. The canonical methionine 392 of matrix metalloproteinase 2 (gelatinase A) is not required for catalytic efficiency or structural integrity: probing the role of the methionine-turn in the metzincin metalloprotease superfamily. J. Biol. Chem. 279, 15615–15620 (2004).
Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).
Chan, Q.W., Howes, C.G. & Foster, L.J. Quantitative comparison of caste differences in honeybee hemolymph. Mol. Cell. Proteomics 5, 2252–2262 (2006).
Han, D.K., Eng, J., Zhou, H. & Aebersold, R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 19, 946–951 (2001).
Li, X.J., Zhang, H., Ranish, J.A. & Aebersold, R. Automated statistical analysis of protein abundance ratios from data generated by stable-isotope dilution and tandem mass spectrometry. Anal. Chem. 75, 6648–6657 (2003).
Fujimoto, N. et al. Extracellular matrix protein 1 inhibits the activity of matrix metalloproteinase 9 through high-affinity protein/protein interactions. Exp. Dermatol. 15, 300–307 (2006).
Starr, A.E. & Overall, C.M. Chapter 13. Characterizing proteolytic processing of chemokines by mass spectrometry, biochemistry, neo-epitope antibodies and functional assays. Methods Enzymol. 461, 281–307 (2009).
Kleifeld, O., Doucet, A., Kizhakkedathu, J. & Overall, C.M. System-wide proteomic identification of protease cleavage products by terminal amine isotopic labeling of substrates. Nat. Protoc. advance online publication, doi:10.1038/nprot.2010.30 (7 March 2010).
Acknowledgements
O.K. was supported by the Centre for Blood Research (CBR) (University of British Columbia), Canadian Institute for Health Research/Heart and Stroke Foundation of Canada (CIHR/HSFC) Strategic Training Program in Transfusion Science research fellowship. A.D. acknowledges the Fonds Quebecois de la Recherche sur la Nature et les Technologies and the Michael Smith Foundation for Health Research (MSFHR) for research fellowships. U.a.d.K. was supported by a Deutsche Forschungsgemeinschaft (DFG) research fellowship. A.P. acknowledges the support from the CBR Strategic Training Program in Transfusion Science. O.S. acknowledges support from the DFG and the MSFHR. A.E.S. is supported by Natural Sciences and Engineering Research Council of Canada, the MSFHR and CIHR Strategic Training Program STP-53877. L.J.F. is the Canada Research Chair in Organelle Proteomics, a Michael Smith Foundation Scholar and a Peter Wall Institute for Advanced Studies Early Career Scholar. J.N.K. is the recipient of a Canadian Blood Services (CBS)/CIHR new investigator award in transfusion science. C.M.O. is supported by a Canada Research Chair in Metalloproteinase Proteomics and Systems Biology. This work was supported by grants from the CIHR and from a program project grant in Breast Cancer Metastases from the Canadian Breast Cancer Research Alliance with funds from the Canadian Breast Cancer Foundation and The Cancer Research Society as well as with an Infrastructure Grant from the Canada Foundation for Innovation (CFI) and the MSFHR. We thank W. Chen and the UBC Centre for Blood Research Mass Spectrometry Suite (supported by the CFI and the MSFHR) for proteomics analysis, G. Butler for analysis of cyclophilin A cleavage by MMP-2 and V. Goebeler for helpful suggestions regarding endoplasmin assays. D.E. Brooks is thanked for his encouragement and use of facilities. The authors thank the LMB Macromolecular Hub at the UBC Centre for Blood Research for the use of their research facilities, which is supported by the CFI and the MSFHR. MCF7 cells, stably transfected with either full-length human MMP-11 or inactive control (MMP-11 (E216A)) were a kind gift from B. Mari (University of Nice, France). Recombinant human proBMP-1, human fibulin-2, human ECM-1a (truncation of 123 amino acid at the N-terminus), mouse sulfated glycoprotein 1 and DPPIV were kindly provided by S. Walter (Johannes Gutenburg University), T. Sasaki (Oregon Health and Science University), J. Merregaert (University of Antwerp), S. Koochekpour (LSU-Health Sciences Center) and C. McIntosh (University of British Columbia), respectively.
Author information
Authors and Affiliations
Contributions
O.K. participated in the project design, developed TAILS, performed proof of principal and MMP-2 experiments, did the bioinformatics data analysis and drafted the manuscript. A.D. participated in the project design, performed TAILS on GluC, MMP-2 and BALF and in vitro assays and revised the manuscript. U.a.d.K. and A.P. participated in the project design, performed the MMP-11 analyses and revised the manuscript. O.S. participated in the project design and the bioinformatics data analysis. A.S. performed the LIX and DPPIV experiments. R.K.K. produced the HPG-ALD polymers. L.J.F. performed the mass spectrometry analyses on his Orbitrap mass spectrometer. J.N.K. is the senior author for the HPG-ALD chemistry and synthesis. He participated in the project design, engineered the HPG-ALD polymer series and participated in the manuscript writing. C.M.O. is the overall senior author for the degradomics and proteomics section of the manuscript. He conceived the project and design and was responsible for project supervision, data interpretation and manuscript writing and provided grant support.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Text and Figures
Supplementary Discussion and Supplementary Results 1–10 (PDF 33721 kb)
Rights and permissions
About this article
Cite this article
Kleifeld, O., Doucet, A., auf dem Keller, U. et al. Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat Biotechnol 28, 281–288 (2010). https://doi.org/10.1038/nbt.1611
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nbt.1611
This article is cited by
-
A novel, robust peptidyl-lys metalloendopeptidase from Trametes coccinea recombinantly expressed in Komagataella phaffii
Applied Microbiology and Biotechnology (2024)
-
Design of a mucin-selective protease for targeted degradation of cancer-associated mucins
Nature Biotechnology (2024)
-
Membrane procoagulation and N‑terminomics/TAILS profiling in Montreal platelet syndrome kindred with VWF p.V1316M mutation
Communications Medicine (2023)
-
The roles of intracellular proteolysis in cardiac ischemia–reperfusion injury
Basic Research in Cardiology (2023)