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A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry.

Proceedings of the National Academy of Sciences of the United States of America (1999-06-23)
T Keough, R S Youngquist, M P Lacey
ABSTRACT

A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.

MATERIALS
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Brand
Product Description

Sigma-Aldrich
Insulin Chain B Oxidized from bovine pancreas, ≥80% (HPLC), powder
Sigma-Aldrich
Chlorosulfonylacetyl chloride, 95%