Molecular mass determination by Electrospray Mass Spectrometry of human pepsins, gastricsin, and porcine pepsin A variants

Proteins are normally analysed by Electrospray Mass Spectrometry (ESMS) as their multiply charged positive ions, when their molecular masses are typically measured to within 0.01% of the values calculated from the primary sequences. The aspartic proteinases obtained from gastric secretions, however, have an insufficient number of basic amino acids to be analysed in this way, but should be well suited to negative ion analysis because they contain a large number of acidic residues. The amount of available data on the analysis of acidic proteins by ESMS is sparse and in order to assess the efficacy of negative ion ESMS, a commercial sample of porcine pepsin A and several proteinases isolated from human gastric juice were analysed and their measured molecular masses compared with values calculated from cDNA sequences (1–3). Attempts were made from mass data, to characterise purified human pepsin A variants with the published cDNA sequences(2); and particular emphasis was placed on obtaining a true molecular mass for human pepsin 1 (fast moving pepsin) for which there is evidence of carbohydrate attachment(4).