title: Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
authors.forename: Alain
authors.initials: A
authors.lastname: Doucet
authors.forename: Anna
authors.initials: A
authors.lastname: Prudova
authors.forename: Ulrich
authors.initials: U
authors.lastname: auf dem Keller
authors.forename: Magda
authors.initials: M
authors.lastname: Gioia
authors.forename: Jayachandran N
authors.initials: JN
authors.lastname: Kizhakkedathu
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D018929:Cell Culture Techniques; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D030562:Databases, Protein; D007553:Isotope Labeling / Q000379:methods*; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome; D020539:Sequence Analysis, Protein / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D011108:Polymers; D011506:Proteins; D020543:Proteome; D010447:Peptide Hydrolases