Derivatization procedures to facilitate de novo sequencing of lysine-terminated tryptic peptides using postsource decay matrix-assisted laser desorption/ionization mass spectrometry

Guanidination of the E-amino group of lysine-terminated tryptic peptides ca n be accomplished selectively in one step with 0-methylisourea hydrogen sul fate. This reaction converts lysine residues into more basic homoarginine r esidues. It also protects the E-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-t ermini of tryptic peptides, The combined reactions convert lysine-terminate d tryptic peptides into modified peptides that are suitable for de novo seq uencing by postsource decay matrix-assisted laser desorption/ionization (MA LDI) mass spectrometry. The guanidination reaction is very pH dependent. Pr oduct yields and reaction kinetics were studied in aqueous solution using e ither NaOH or diisopropylethylamine as the base. Methods are reported for d erivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels, The postsource decay (PSD) MALDI tandem mass spectra of a model pep tide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine a nalog are compared. These spectra show the influence that each chemical mod ification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI seque ncing of derivatized peptides obtained from solution digests of model prote ins and from in-gel digests of 2D-gel separated proteins. Copyright (C) 200 0 John Wiley & Sons, Ltd.