Characterisation of variant forms of prophenin: mechanistic aspects of the fragmentation of proline-rich peptides.
Prophenin 1 (PF-1) is a 79-residue polypeptide originally isolated from porcine leukocytes. Its amino acid sequence has been determined by a combination of mass spectrometry and Edman degradation (Harwig SSL. et al. FEBS Lett. 1995; 362: 65). Prophenin (PF) and variants thereof are also found in organic extracts of porcine pulmonary tissue (Wang Y. et al. FEBS Lett. 1999; 460: 257). In the present study we have characterised the variant forms of PF found in these extracts using nano-electrospray (nano-ES) high resolution and tandem mass spectrometry. The major forms of PF found in these extracts by nano-ES mass spectrometry are the 80-residue polypeptides prophenin-2-Pyr (PF-2-Pyr) and prophenin-2-Gln (PF-2-Gln). Prophenin-2-Pyr is refractory to Edman degradation due to the presence of an N-terminal pyroglutamic residue. In PF-2-Gln the N-terminal residue is glutamine and the C-terminus is amidated. In porcine pulmonary extracts PF-1 is present to only a minor extent. Other shorter polypeptides are also found in these extracts including 18- and 17-residue C-terminal fragments of PF. The primary structure of PF is highly unusual in that it shows four almost perfect decamer repeats of FPPPN(V/F)PGPR and, out of the 79/80 residues, 42 are proline and 14 are phenylalanine. Tryptic digestion of PF gives peptides containing the decamer repeat and collision-induced dissociation of these peptides provides an insight into the fragmentation mechanisms of proline-rich peptides. Facile cleavage within the Pro-Pro-Pro sequence of these peptides suggests the involvement of a cyclic peptide in the fragmentation mechanism. Fragmentation mechanisms that account for the formation of fragment ions at other cleavage sites are also discussed.
Wang
Y
Y
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.
Johansson
J
J
Griffiths
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry*; D023181:Antimicrobial Cationic Peptides; D008168:Lung / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D011392:Proline / Q000032:analysis*; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D010446:Peptide Fragments; D011506:Proteins; C092764:prophenin 1; D011392:Proline