Post-translational modifications in aspartate aminotransferase from Sulfolobus solfataricus. Detection of N-epsilon-methyllysines by mass spectrometry.
Advanced mass spectrometric procedures have been extensively used to provide an accurate structural characterization of aspartate aminotransferase from Sulfolobus solfataricus. The amino acid sequence of this enzyme had previously been deduced from the DNA sequence. The accurate molecular mass of the protein, determined using electrospray mass spectrometry, demonstrated that the amino acid sequence deduced was correct and ruled out the possible presence of large covalent modifications which had been postulated to fit the much higher molecular mass obtained from previous SDS/PAGE experiments. The definition of the entire primary structure of aspartate aminotransferase from S. solfataricus was achieved by exploiting a new mass spectrometric mapping strategy. Initially, the molecular mass of relatively large protein fragments produced by CNBr hydrolysis was accurately determined using electrospray mass spectrometry. The protein regions where structural modifications had occurred were easily identified from their anomalous mass values. The corresponding CNBr fragments were then subdigested with suitable proteases and the resulting peptide mixtures were analysed by fast-atom-bombardment mass spectrometry. This mapping approach led to the detection of two partially modified lysine residues at positions 202 and 384, which had been converted to their N-epsilon-methyl derivatives to a substoichiometric extent.
Zappacosta
F
F
Servizio di Spettrometria di Massa del CNR, Napoli, Italy.
Sannia
G
G
Savoy
L A
LA
Marino
G
G
Pucci
P
P
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D016944:Sulfolobus / Q000201:enzymology*
C005205:epsilon-N-methyllysine; D001219:Aspartate Aminotransferases; D008239:Lysine; D003488:Cyanogen Bromide