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 com plex of the pea leaf mitochondria, was investigated by liquid chromato graphy/mass spectromety (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using both continuous flow fast atom bombardm ent (CF-FAB) and electrospray ionization (ESI) mass spectrometry. Dete rmination of the molecular weight of the entire protein, a 14 kDa subu nit of the glycine decarboxylase complex, was achieved by ESI mass spe ctrometry and revealed covalent binding of the protein to the stabiliz ing agent beta-mercapto-ethanoL On-line LC/MS analysis of peptides ari sing from the endoproteinase Glu-C digestion of the H-protein was achi eved using capillary columns (0.25 mm i.d.), and permitted confirmatio n of the previously reported sequence deduced from cDNA cloning experi ments. 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 aci d cofactor, a rather unusual modified lysine residue which interacts w ith different active sites in the enzyme complex, was achieved using b oth LC/CF-FAB-MS and LC/ESI-MS. The exact location of this modified ly sine residue was determined by obtaining fragment spectra of multiply protonated precursor ions of selected peptides, using on-line LC/MS/MS techniques.