Abstract
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.
Highlights
Selective enzymatic biotinylation of N terminiC-terminal amine isotope labeling of substrates (TAILS) (ϩ) Positive selection of unmodified N termini (ϩ) Does not require chemical modification (Ϫ) Requires expensive patent protected enzyme (Ϫ) Requires large amounts of sample (Ϫ) Enriched peptides are shortened by one residue (Ϫ) Not compatible with chemical stable-isotope labeling (ϩ) Negative selection of N and C termini (Ϫ) Extensive fractionation enhances sample loss (Ϫ) Ͼ50 fractions/sample, making it very instrument intensive (Ϫ) Loss of His- and Arg-containing peptides during strong cation exchange chromatography (ϩ) Negative selection of modified and unmodified N-termini (ϩ) Very low nonspecific binding to polymer (Ϫ) Requires commercially available hyperbranched polyglycerol aldehyde polymer (ϩ) Negative selection of modified and unmodified N termini (Ϫ) Loss of phosphorylated N-terminal peptides (Ϫ) Losses due to nonspecific binding to TiO2 material (ϩ) Negative selection of modified N termini (Ϫ) No retention of unmodified N termini (Ϫ) Loss of His-containing peptides (ϩ) Negative selection of modified and unmodified C termini (ϩ) Chemical tag identifies unmodified C termini (Ϫ) Difficult to achieve complete labeling of carboxyl groups a Published name or description of enrichment method
Proteolysis involves the breakdown of proteins into smaller polypeptides or amino acids through the hydrolysis of peptide bonds by a protease
Two methods currently exist for proteomic analysis of protein C termini: C-terminal amine-based isotope labeling of substrates (C-terminal amine isotope labeling of substrates (TAILS)), and COFRADIC combined with strong cation exchange chromatography [54, 55] (Table I)
Summary
C-TAILS (ϩ) Positive selection of unmodified N termini (ϩ) Does not require chemical modification (Ϫ) Requires expensive patent protected enzyme (Ϫ) Requires large amounts of sample (Ϫ) Enriched peptides are shortened by one residue (Ϫ) Not compatible with chemical stable-isotope labeling (ϩ) Negative selection of N and C termini (Ϫ) Extensive fractionation enhances sample loss (Ϫ) Ͼ50 fractions/sample, making it very instrument intensive (Ϫ) Loss of His- and Arg-containing peptides during strong cation exchange chromatography (ϩ) Negative selection of modified and unmodified N-termini (ϩ) Very low nonspecific binding to polymer (Ϫ) Requires commercially available hyperbranched polyglycerol aldehyde polymer (ϩ) Negative selection of modified and unmodified N termini (Ϫ) Loss of phosphorylated N-terminal peptides (Ϫ) Losses due to nonspecific binding to TiO2 material (ϩ) Negative selection of modified N termini (Ϫ) No retention of unmodified N termini (Ϫ) Loss of His-containing peptides (ϩ) Negative selection of modified and unmodified C termini (ϩ) Chemical tag identifies unmodified C termini (Ϫ) Difficult to achieve complete labeling of carboxyl groups a Published name or description of enrichment method. SILAC, stable isotope labeling by amino acids in cell culture
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