Liquid chromatographic–high-resolution mass spectrometric and tandem mass spectrometric identification of synthetic peptides using electrospray ionization

Abstract Liquid chromatography–high-resolution electrospray mass spectrometry (LC–ESI-MS) was investigated for the identification of known and unknown synthetic peptides in a research effort designed to evaluate the applicability of this and complementary MS techniques for peptide characterization and identification. The monoisotopic molecular masses of five related peptides with molecular masses between 2000 and 2500 u were acquired with a resolution of 3000 (10% valley). Under narrow and wide mass range magnetic sector scanning conditions monoisotopic molecular mass errors were typically in the 10–20 and 30–40 ppm range, respectively. Tryptic maps were generated for each peptide following LC–ESI-MS analysis and collisionally activated dissociation (CAD) in the ESI interface resulted in the production of characteristic product ions that enabled amino acid sequencing of the tryptic fragments. Unknown identification was demonstrated during analysis of an incomplete synthetic peptide reaction mixture. The synthesis of an 18 amino acid peptide, LTTAVKKVLTTGLPALIS, was not successful. In its place were six unknown peptides that were identified on the basis of monoisotopic molecular mass and amino acid sequence data. The monoisotopic molecular masses of these unknowns were determined to within 10–20 ppm with a resolution of 3500 (10% valley). Amino acid sequences for the six peptides were generated during ESI–MS–MS analysis. Finally two synthetic peptides differing only by the incorporation of a 13 C at leucine were analysed with a resolution of 6000 (10% valley) to confirm that the isotopic distributions were consistent with theoretical expectations.