De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/collision-induced dissociation mass spectrometry.
A series of synthetic peptides (3-15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
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I
Lindh
L
L
Hjelmqvist
T
T
Bergman
J
J
Sjövall
W J
WJ
Griffiths
D000444:Aldehyde Dehydrogenase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D006224:Cricetinae; D004563:Electrochemistry; D010450:Endopeptidases / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D008647:Mesocricetus; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis* / Q000737:chemistry
D010446:Peptide Fragments; D010455:Peptides; D001224:Aspartic Acid; D018698:Glutamic Acid; D000444:Aldehyde Dehydrogenase; D010450:Endopeptidases