Gas chromatographic mass spectrometric sequencing of peptides and proteins containing gamma-carboxyglutamic acid

A generally applicable strategy has been developed for the primary sequence determination of peptides and proteins containing gamma-carboxyglutamic acid. The intact peptide or protein is either dissolved in, or allowed to come into vapor phase contact with, 0.05 M DCI and then heated in vacuo at 110 degrees C for several hours. Under these conditions gamma-carboxyglutamic acid quantitatively decarboxylates and incorporates two atoms of deuterium per molecule, resulting in the formation of gamma-dideuteroglutamyl residues. Following enzymatic or acidic degradation of the protein the peptide mixture generated is converted (without further isolation of individual peptides) to the N-trifluoroacetylated, O-trimethylsilylated polyamino alcohols and subsequently analyzed by gas chromatography mass spectrometry. Peptide fragments in which gamma-carboxyglutamic acid was present show sequence ions in their mass spectra corresponding to those of glutamic acid, but shifted upwards by 2 amu. This approach has been used to identify the positions of Gla in a tryptic peptide isolated from blood coagulation factor IX, and is currently being employed in the sequence determination of the Gla-containing bone protein osteocalcin.