Sequencing branched peptides with CID/PSD MALDI-TOF in the low-picomole range: application to the structural study of the posttranslational polyglycylation of tubulin.
Sequencing conditions for postsource decay and collision-induced dissociation/postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been optimized to elucidate the structure of polyglycylation of tubulin. This posttranslational modification involves the linkage of multiple glycine residues through the gamma-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual alpha- and beta-tubulin polypeptides contain respectively three and four potential glycylation sites. The sample preparation we used was the thin-layer preparation of the target specimen in the presence of alpha-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence DATAEEEGEFEEEGEQ) shows that the peptides fragment regularly to form major fragments of b- and y-type ions with negligible side-chain fragmentation. The rules were applied to the structural elucidation of a Paramecium beta-tubulin hexaglycylated peptide available in the subpicomole range. Polyglycylation was identified on the last four glutamic acid residues.
Vinh
J
J
Laboratoire de Neurobiologie, Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris, CNRS URA 2054, France. joelle.vinh@espci.fr
Loyaux
D
D
Redeker
V
V
Rossier
J
J
D000595:Amino Acid Sequence; D000818:Animals; D005998:Glycine / Q000737:chemistry; D008832:Microchemistry; D008969:Molecular Sequence Data; D010247:Paramecium; D010449:Peptide Mapping / Q000379:methods; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014404:Tubulin / Q000737:chemistry*
D015800:Protozoan Proteins; D014404:Tubulin; D005998:Glycine