Journal of Chromatography, 646 (1993) 175-184 Elsevier Science Publishers B.V., Amsterdam CHROMSYMP. 2867 Analytical methods for differentiating minor sequence variations in related peptides Paul A. Grieve* International Food Institute of Queensland, 19 Hercules Street, Hamilton, Qld. 4007 (Australia) Alun Jones and Paul F. Alewood Centre for Drug Design and Development, University of Queensland, St. Lucia, Qld. 4067 (Australia) ABSTRACT 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 Bat-X) was determined by ionspray MS to be 5149a0.5. 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. Bat-X had similar characteristics to Bat-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 Bat-X produced fragments with masses (M, 785 and 4224 and 3100 respectively) consistent with those expected from a peptide with the reported sequence of Bat-5. Bat-X differed from Bat-5 in the number of amino acid residues (43 for Bat-X versus 42 for Bat-5) and contained glycine which Bat-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, 785 tryptic fragment of Bat-X, showed that this peptide contained a six residue sequence (RFPPIR-) not found in Bat-5 which, based on its reported sequence, contained a M, 785 tryptic fragment with the sequence -FRPPIR-. This difference in sequence of Bat-X compared with Bat-5 illustrates the application of electrospray (ionspray) MS techniques to the detection and identification of minor differences in related protein/peptide forms. INTRODUCTION Multicellular organisms contain specialised cells (granulocytes, macrophages, cytotoxic lymphocytes, natural killer cells etc.) that can selectively destroy invading organisms such as bacteria. Such cells, including the bovine neu- trophil, contain a variety of lytic enzymes and antimicrobial/cytotoxic proteins stored in cyto- plasmic granules [ 11. The term “bactenecin” has been ascribed to antibacterial proteins/peptides isolated from * Corresponding author. bovine neutrophils. To date three bactenecins have been isolated from the large granules of the bovine neutrophil and fully characterised. These include a defensin-like, cyclic dodecapeptide [2] and two unique proline- and arginine-rich pep- tides Bat-5 and Bat-7 which do not resemble the reported sequences of other mammalian anti- microbial peptides [3]. The two bactenecins Bat-5 and Bat-7 (A4,= XKKI and ~7000, respectively) were character- ised by their limited number of amino acid types, including a high proline (~45%) and arginine (>23%) content and hydrophobic amino acids. Bat-5 and Bat-7 are highly antibacterial towards Gram-negative organisms (Eschetichiu COU) but 0021-9673/93/$06.00 @ 1993 Elsevier Science Publishers B.V. All rights reserved