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

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Abstract
A generally applicable strategy has been developed for the primary sequence determination of peptides and proteins containing γ‐carboxyglutamic acid. The intact peptide or protein is either dissolved in, or allowed to come into vapor phase contact with, 0.05 M DCl and then heated in vacuo at 110°C for several hours. Under these conditions γ‐carboxyglutamic acid quantitatively decarboxylates and incorporates two atoms of deuterium per molecule, resulting in the formation of γ‐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 γ‐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.

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