Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry.
In hot electron capture dissociation (HECD), multiply protonated polypeptides fragment upon capturing approximately 11-eV electrons. The excess of energy upon the primary c, z* cleavage induces secondary fragmentation in z* fragments. The resultant w ions allow one to distinguish between the isomeric Ile and Leu residues. The analytical utility of HECD is evaluated using tryptic peptides from the bovine milk protein PP3 containing totally 135 amino acid residues. Using a formal procedure for Ile/Leu (Xle) residue assignment, the identities of 20 out of 25 Xle residues (80%) were determined. The identity of an additional two residues could be correctly guessed from the absence of the alternative w ions, and only two residues, for which neither expected nor alternative w ions were observed, remained unassigned. Reinspection of conventional ECD spectra also revealed the presence of Xle w ions, although at lower abundances, with 44% of all Xle residues distinguished. Using a dispenser cathode as an electron source, identification of four out of five Xle residues in a 2.7-kDa peptide was possible with one acquisition 2 s long, with identification of all five residues by averaging of five such acquisitions. Unlike the case of high-energy collision-induced dissociation, no d ions were observed in the HECD of tryptic peptides.
Kjeldsen
Frank
F
Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Haselmann
Kim F
KF
Sørensen
Esben S
ES
Zubarev
Roman A
RA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D017357:Cyclotrons; D006023:Glycoproteins / Q000737:chemistry*; D007532:Isoleucine / Q000032:analysis*; D007930:Leucine / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D008894:Milk Proteins / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D020539:Sequence Analysis, Protein / Q000379:methods*
D000596:Amino Acids; D002364:Caseins; D006023:Glycoproteins; D008894:Milk Proteins; C085522:PP3 protein, Bos taurus; D010446:Peptide Fragments; D007532:Isoleucine; D007930:Leucine