Analytical methods for differentiating minor sequence variations in related peptides

A proline-rich peptide was isolated and purified to homogeneity from an extract of bovine neutrophil granules using semi-preparative RP-HPLC. The relative molecular mass of the peptide (called Bac-X) was determined by ionspray MS to be 5149 +/- 0.5. The amino acid composition of the peptide was characterized by its limited number of amino acid types, which included a high proline (43.3%) and arginine content (20.3%), and hydrophobic residues. Bac-X had similar characteristics to Bac-5, a previously characterised bactenecin of bovine neutrophil granules, with respect to its proline, arginine and hydrophobic amino acid content, molecular mass and antibacterial specificity. Tryptic and N-bromosuccinimide digestion of Bac-X produced fragments with masses (M(r) 785 and 4224 and 3100 respectively) consistent with those expected from a peptide with the reported sequence of Bac-5. Bac-X differed from Bac-5 in the number of amino acid residues (43 for Bac-X versus 42 for Bac-5) and contained glycine which Bac-5 did not. However, the calculated molecular mass of the peptide, based on the amino acid compositional data, did not match the experimental value. The purified peptide could not be sequenced by Edman degradation due to apparent blockage of the N-terminus. Partial sequence information, obtained by LC-MS and collision induced dissociation MS-MS analysis of a M(r) 785 tryptic fragment of Bac-X, showed that this peptide contained a six residue sequence (-RFPPIR-) not found in Bac-5 which, based on its reported sequence, contained a M(r) 785 tryptic fragment with the sequence -FRPPIR-. This difference in sequence of Bac-X compared with Bac-5 illustrates the application of electrospray (ionspray) MS techniques to the detection and identification of minor differences in related protein/peptide forms.