Structural elucidation of N-terminal post-translational modifications by mass spectrometry: Application to chicken enolase and the α- and β-subunits of bovine mitochondrial F 1-ATPase

Abstract

Peptides generated from enzymatic hydrolysis of chicken enolase and the α- and β-subunits of bovine F 1-ATPase were analyzed by mass spectrometry to determine the nature of their modified N-termini. In the case of chicken enolase, a peptide was isolated from a Staphlococcus aureus proteinase digest by HPLC and analyzed directly by fast atom bombardment mass spectrometry (FABMS). In conjunction with mass spectral evidence obtained from the methyl ester derivative and a secondary tryptic peptide, a structure is proposed containing an N-acetyl serine at the N-terminus. The α-subunit of bovine mitochondrial ATPase was chromatographed by HPLC after S. aureus proteinase digestion and a single peak was analyzed on the basis of predicted retention times. A M r 716 was determined by FABMS and pyrrolidone carboxylic acid was deduced on the basis of its amino acid composition and partial Edman sequence data. The β-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the α 2 β 2 complex. These peptides were analyzed by both Edman degradation and FABMS. These data showed the N-terminus to be frayed with N-terminal sequences beginning in pyro-Glu-Ala-Ser, Gln-Ala-Ser, Glu-Ala-Ser, Ala-Ser, and Ser but with no N-acetyl-Ser as was previously thought.