Sequencing Branched Peptides with CID/PSD MALDI-TOF in the Low-Picomole Range: Application to the Structural Study of the Posttranslational Polyglycylation of Tubulin

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Abstract
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 γ-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual α- and β-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 α-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence 427DATAEEEGEFEEEGEQ441) 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 β-tubulin hexaglycylated peptide available in the subpicomole range. Polyglycylation was identified on the last four glutamic acid residues.

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