Sequence analysis of derivatized peptides by high-performance liquid chromatography-mass spectrometry.
N-Acetyl-N,O,S-permethylated derivatives of oligopeptides were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using a moving belt interface. A heated-gas nebulizer was employed for sample deposition, thus permitting the effective use of a water-methanol gradient covering the range from 5% to 95% water at mobile phase flow-rates of 0.5 ml/min. We demonstrate in this paper that it is possible to sequence the octapeptide derived from the C-chain of glucagon by HPLC-MS analysis of a permethylated enzymatic hydrolysate of this peptide using overlap information from the mass spectral patterns. Moreover, it is shown that peptides not readily amenable to analysis by gas chromatography-MS can be analyzed using this approach. Preliminary results suggest that N-acetyl-methyl ester derivatives of oligopeptides may in specific cases also be a useful alternative for HPLC-MS analysis of complex oligopeptide mixtures.
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002918:Chymotrypsin; D004744:Enkephalin, Methionine / Q000032:analysis; D005934:Glucagon / Q000032:analysis; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D009842:Oligopeptides / Q000032:analysis; D010455:Peptides / Q000032:analysis*
D009842:Oligopeptides; D010455:Peptides; D004744:Enkephalin, Methionine; D005934:Glucagon; D002918:Chymotrypsin