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Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Nature Protocols volume 6, pages 1578–1611 (2011)Cite this article

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Abstract

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo–N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.

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Figure 2: Peptide relative quantification with dimethylation, SILAC or iTRAQ isotopic-labeling strategies.
Figure 3: Schematic representation of bioinformatics analysis pipeline for TAILS data.
Figure 1: Schematic representation of the TAILS workflow.
Figure 4: Schematic representation of the immobilization procedure for a single piece of Empore material on a pipette tip.

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Author notes

  1. Oded Kleifeld, Ulrich auf dem Keller & Magda Gioia

    Present address: Present addresses: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia (O.K.); Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada (A.D); Institute of Cell Biology ETH Zurich, Zurich, Switzerland (U.a.d.K.); Department of Experimental Medicine and Biochemical Sciences, Università di Roma Tor Vergata, Roma, Italy (M.G.).,

  2. Oded Kleifeld, Alain Doucet, Anna Prudova, Ulrich auf dem Keller and Magda Gioia: These authors contributed equally to this work.

  3. chris.overall@ubc.ca

Authors and Affiliations

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

    Oded Kleifeld, Alain Doucet, Anna Prudova, Ulrich auf dem Keller, Magda Gioia & Christopher M Overall

  2. Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada

    Oded Kleifeld, Alain Doucet, Anna Prudova, Ulrich auf dem Keller, Magda Gioia & Christopher M Overall

  3. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada

    Jayachandran N Kizhakkedathu

  4. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada

    Jayachandran N Kizhakkedathu

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Contributions

O.K. developed dimethylation-TAILS and drafted the manuscript. A.D. participated in the development and optimization of dimethylation-TAILS and participated in the manuscript writing. A.P. developed iTRAQ-TAILS and revised the manuscript. U.a.d.K. participated in the development of analysis tools of iTRAQ-TAILS, wrote TAILS-ANNOTATOR and revised the manuscript. M.G. developed SILAC-TAILS and revised the manuscript. J.N.K. engineered the HPG-ALD polymer series and participated in methods development and manuscript writing. C.M.O. conceived the TAILS concept, projects and design, and was responsible for project supervision, data interpretation, manuscript writing and providing grant support.

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Kleifeld, O., Doucet, A., Prudova, A. et al. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates. Nat Protoc 6, 1578–1611 (2011). https://doi.org/10.1038/nprot.2011.382

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