title: Structural elucidation of N-terminal post-translational modifications by mass spectrometry: application to chicken enolase and the alpha- and beta-subunits of bovine mitochondrial F1-ATPase.
abstract: Peptides generated from enzymatic hydrolysis of chicken enolase and the alpha- and beta-subunits of bovine F1-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 Staphylococcus 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 alpha-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 Mr 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 beta-subunit of ATPase produced a series of closely eluting peaks on HPLC after limited digestion with trypsin of the alpha 2 beta 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.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D008928:Mitochondria / Q000201:enzymology*; D010455:Peptides / Q000032:analysis; D010751:Phosphopyruvate Hydratase / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000032:analysis*
chemical_list: D010455:Peptides; D006180:Proton-Translocating ATPases; D010751:Phosphopyruvate Hydratase