Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.
Marekov
Lyuben N
LN
Laboratory of Skin Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. marekovl@mail.nih.gov
Steinert
Peter M
PM
D000595:Amino Acid Sequence; D001557:Benzenesulfonates / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D001557:Benzenesulfonates; D002135:Calcium-Binding Proteins; D017879:Isothiocyanates; D010446:Peptide Fragments; C015308:4-isothiocyanatobenzenesulfonate; C498712:TGM3 protein, human; D011503:Transglutaminases; D014357:Trypsin