Letter | Published:

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products

Nature Biotechnology volume 28, pages 281288 (2010) | Download Citation


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.

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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

Author notes

    • Oded Kleifeld
    • , Ulrich auf dem Keller
    •  & Oliver Schilling

    Present addresses: Israel Institute of Technology, Faculty of Biology, Haifa, Israel (O.K.); Institute of Cell Biology, ETH Zürich, Zürich, Switzerland (U.a.d.K.), University of Freiburg, Institute for Molecular Medicine and Cell Research, Freiburg, Germany (O.S.).

    • Oded Kleifeld
    •  & Alain Doucet

    These authors contributed equally to this work.


  1. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.

    • Oded Kleifeld
    • , Alain Doucet
    • , Ulrich auf dem Keller
    • , Anna Prudova
    • , Oliver Schilling
    • , Amanda E Starr
    • , Leonard J Foster
    •  & Christopher M Overall
  2. Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

    • Oded Kleifeld
    • , Alain Doucet
    • , Ulrich auf dem Keller
    • , Anna Prudova
    • , Oliver Schilling
    •  & Christopher M Overall
  3. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.

    • Rajesh K Kainthan
    •  & Jayachandran N Kizhakkedathu
  4. Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.

    • Oded Kleifeld
    • , Alain Doucet
    • , Ulrich auf dem Keller
    • , Anna Prudova
    • , Oliver Schilling
    • , Rajesh K Kainthan
    • , Amanda E Starr
    • , Jayachandran N Kizhakkedathu
    •  & Christopher M Overall


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Jayachandran N Kizhakkedathu or Christopher M Overall.

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