title: Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) N-terminal sequencing of proteins and proteolytic cleavage sites by quantitative mass spectrometry.
abstract: Edman degradation is a long-established technique for N-terminal sequencing of proteins and cleavage fragments. However, for accurate data analysis and amino acid assignments, Edman sequencing proceeds on samples of single proteins only and so lacks high-throughput capabilities. We describe a new method for the high-throughput determination of N-terminal sequences of multiple protein fragments in solution. Proteolytic processing can change the activity of bioactive proteins and also reveal cryptic binding sites and generate proteins with new functions (neoproteins) not found in the parent molecule. For example, extracellular matrix (ECM) protein processing often produces multiple proteolytic fragments with the generation of cryptic binding sites and neoproteins by ECM protein processing being well documented. The exact proteolytic cleavage sites need to be identified to fully understand the functions of the cleavage fragments and biological roles of proteases in vivo. However, the identification of cleavage sites in complex high molecular proteins such as those composing the ECM is not trivial. N-terminal microsequencing of proteolytic fragments is the usual method employed, but it suffers from poor resolution of sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and is inefficient at identifying multiple cleavages, requiring preparation of numerous gels or membrane slices for analysis. We recently developed Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS) to overcome these limitations as a complement for N-terminal sequencing. ATOMS employs isotopic labeling and quantitative tandem mass spectrometry to identify cleavage sites in a fast and accurate manner. We successfully used ATOMS to identify nearly 100 cleavage sites in the ECM proteins laminin and fibronectin. Presented herein is the detailed step-by-step protocol for ATOMS.
authors.affiliation: Centre for Blood Research, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Alain
authors.initials: A
authors.lastname: Doucet
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007797:Laminin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D005353:Fibronectins; D007797:Laminin; D010446:Peptide Fragments; D014357:Trypsin