"De novo" peptide sequencing by MALDI-quadrupole-ion trap mass spectrometry: a preliminary study.
Collision-induced dissociation of singly charged peptide ions produced by resonant excitation in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer yields relatively low complexity MS/MS spectra that exhibit highly preferential fragmentation, typically occurring adjacent to aspartyl, glutamyl, and prolyl residues. Although these spectra have proven to be of considerable utility for database-driven protein identification, they have generally been considered to contain insufficient information to be useful for extensive de novo sequencing. Here, we report a procedure for de novo sequencing of peptides that uses MS/MS data generated by an in-house assembled MALDI-quadrupole-ion trap mass spectrometer (Krutchinsky, Kalkum, and Chait Anal. Chem. 2001, 73, 5066-5077). Peptide sequences of up 14 amino acid residues in length have been deduced from digests of proteins separated by SDS-PAGE. Key to the success of the current procedure is an ability to obtain MS/MS spectra with high signal-to-noise ratios and to efficiently detect relatively low abundance fragment ions that result from the less favorable fragmentation pathways. The high signal-to-noise ratio yields sufficiently accurate mass differences to allow unambiguous amino acid sequence assignments (with a few exceptions), and the efficient detection of low abundance fragment ions allows continuous reads through moderately long stretches of sequence. Finally, we show how the aforementioned preferential cleavage property of singly charged ions can be used to facilitate the de novo sequencing process.
Zhang
Wenzhu
W
The Rockefeller University, New York, New York 10021, USA.
Krutchinsky
Andrew N
AN
Chait
Brian T
BT
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D028223:Pinus / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010455:Peptides; D010940:Plant Proteins