Abstract
Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.
Highlights
Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics
ITRAQ Whole Protein Labeling—Here we modified our recently published terminal amine isotopic labeling of substrates (TAILS) protocol [32] with the incorporation of isobaric tag for relative and absolute quantitation (iTRAQ) labeling for the multiplexing of four to eight samples per analysis that allows for more accurate quantification and versatile experimental designs (Fig. 1)
This represents a key step of the new TAILS workflow as it (i) blocks protein N-termini, allowing for their subsequent enrichment by negative selection along with all naturally blocked N-termini, such as those with acetylated or cyclized N-termini [32]; (ii) simultaneously introduces a stable isotope tag to ␣-amino groups of protein N-termini and -amino groups of lysine residues, allowing for accurate quantification; and (iii) because trypsin cannot cleave at iTRAQ-blocked lysine residues, trypsinization yields peptides with Arg-C-like specificity, lengthening most protease-truncated semitryptic neoN-peptides for improved MS/MS identification and substrate coverage
Summary
Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Successful antiproteolytic therapies include those targeting angiotensin convertase in heart disease, dipeptidyl-peptidase IV in diabetes, and human immunodeficiency virus protease-1 in AIDS [12], whereas some, such as matrix metalloproteinase (MMP) inhibitors in cancer, have failed [13, 14] Such drug failures have been attributed to deficiencies in knowledge, limited information on substrate repertoires ( known as substrate degradomes), contributing to the poor understanding of complex protease function in health and disease [7, 15]. Complete annotation of substrates and their cleavage sites is warranted, and this is best done in an unbiased manner on a global scale
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