Characterization of the primary structure of H-protein from Pisum sativum and location of a lipoic acid residue by combined liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry.
A purified extract of H-protein, a subunit of the glycine cleavage complex of the pea leaf mitochondria, was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using both continuous flow fast atom bombardment (CF-FAB) and electrospray ionization (ESI) mass spectrometry. Determination of the molecular weight of the entire protein, a 14 kDa subunit of the glycine decarboxylase complex, was achieved by ESI mass spectrometry and revealed covalent binding of the protein to the stabilizing agent beta-mercapto-ethanol. On-line LC/MS analysis of peptides arising from the endoproteinase Glu-C digestion of the H-protein was achieved using capillary columns (0.25 mm i.d.), and permitted confirmation of the previously reported sequence deduced from cDNA cloning experiments. The detailed interpretation of data extracted from these LC/MS experiments facilitated identification of peptides containing modified amino acid residues. In particular the identification of a lipoic acid cofactor, a rather unusual modified lysine residue which interacts with different active sites in the enzyme complex, was achieved using both LC/CF-FAB-MS and LC/ESI-MS. The exact location of this modified lysine residue was determined by obtaining fragment spectra of multiply protonated precursor ions of selected peptides, using on-line LC/MS/MS techniques.
Mérand
V
V
Institut de Biologie Structurale, Grenoble, France.
Forest
E
E
Gagnon
J
J
Monnet
C
C
Thibault
P
P
Neuburger
M
M
Douce
R
R
D000594:Amino Acid Oxidoreductases; D002352:Carrier Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid; D007887:Fabaceae; D050959:Glycine Decarboxylase Complex; D050961:Glycine Decarboxylase Complex H-Protein; D050841:Glycine Dehydrogenase (Decarboxylating); D013058:Mass Spectrometry; D010944:Plants / Q000737:chemistry*; D010946:Plants, Medicinal; D008063:Thioctic Acid / Q000032:analysis*
D002352:Carrier Proteins; D050961:Glycine Decarboxylase Complex H-Protein; D008063:Thioctic Acid; D000594:Amino Acid Oxidoreductases; D050959:Glycine Decarboxylase Complex; D050841:Glycine Dehydrogenase (Decarboxylating)