Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.
Proteins were digested in normal and highly 18O-enriched water using proteases commonly employed for protein sequencing. The extent of 18O incorporation into the resulting peptide fragments was characterized by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The endoproteinases trypsin, Lys-C and Glu-C incorporate two atoms of 18O, resulting in a mass shift of +4 D for the peptide fragments. This indicates that, following proteolytic cleavage, peptide products continue to interact with these proteases and undergo repeated binding/hydrolysis cycles, resulting in complete equilibration of both oxygens in the carboxy terminus of the fragments with oxygen from solvent water. In contrast, chymotrypsin and Asp-N incorporate only one atom of 18O, resulting in a mass shift of +2 D, indicating that after the cleavage step these proteases do not accept the peptides as substrates. In addition, it was found that the proteases trypsin, Glu-C, and Lys-C exhibit minor or nontypical sequence specificities, resulting in unexpected peptide fragments. These fragments incorporate only one 18O atom, indicating that they do not undergo further binding/hydrolysis cycles with the enzyme. Thus, it is possible to discriminate between enzyme-typical peptide fragments with mass shifts of +4 D and nontypical fragments with mass shifts of only +2 D. Based on these observations, protein digest strategies are described for the generation of 1:1 ion doublets spaced either by 2 or 4 D. In addition, the C-terminus of a protein can be identified by the absence of an ion doublet in the corresponding peptide fragment. In protein sequencing by mass spectrometry, digest protocols generating ion doublets provide the most clear-cut analytical results for the recognition of ion series in ESI-MS/MS and MALDI post-source decay (PSD) product ion spectra. Only the mass spectrometric fragment ions of a C-terminal series show ion doublets spaced either by 2 or 4 D, whereas the fragment ions belonging to an N-terminal series remain unshifted. This assignment unequivocally reveals the direction of the identified sequence.
Schnölzer
M
M
Department of Cell Biology, German Cancer Research Center, Heidelberg, Germany.
Jedrzejewski
P
P
Lehmann
W D
WD
D010450:Endopeptidases / Q000378:metabolism; D004723:Endorphins / Q000032:analysis; D006801:Humans; D006868:Hydrolysis; D007553:Isotope Labeling; D007633:Keratins / Q000032:analysis; D008666:Metalloendopeptidases / Q000378:metabolism; D015394:Molecular Structure; D009496:Neurotensin / Q000032:analysis; D010103:Oxygen Isotopes; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D012697:Serine Endopeptidases / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D004723:Endorphins; D010103:Oxygen Isotopes; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; D009496:Neurotensin; D007633:Keratins; C041128:beta-casomorphin 7; C041968:neurotensin (1-8); D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; C058921:endoproteinase Asp-N; D011761:Pyrrolidonecarboxylic Acid