Analysis of the primary structure and post-translational modifications of the Schistosoma mansoni antigen Smp28 by electrospray mass spectrometry.
The Schistosoma mansoni glutathione-S-transferase with an apparent molecular mass of 28 kDa, Smp28, has a blocked N-terminus which has been elucidated with the aid of the cDNA sequence combined with mass spectrometry and amino acid composition analysis of the N-terminal tryptic peptide. The blocked N-terminal tryptic peptide (m/z 695.8) contained an equimolar ratio of E, G, H, A, I and K3 upon amino acid composition analysis in agreement with its expected sequence AGEHIK, and showed a delta m = +41.7 Da compared to the predicted mass, which is consistent with the N-terminal alanine being acetylated (delta m = +42.0 Da). The mass of the complete molecule (23,744.5 +/- 3.3 Da) determined by electrospray mass spectrometry showed a further mass increase of 14 Da with respect to Smp28 containing an N-acetylated alanine. This result is consistent with one of the seven methionines being present as a methionine sulfoxide in ca. 90% of the Smp28 molecules in this preparation. Tryptic mapping of Smp28 showed five of the seven methionines to be partially oxidized by mass spectrometry. This is indicative of the ease with which this modification occurs. Two minor components were detected along with the intact molecule, corresponding to modified forms of the molecule, originating from reaction of the only cysteine residue either with itself forming a covalent dimer or with glutathione. On-line liquid chromatography-mass spectrometry has been compared with the off-line complete tryptic map of Smp28 confirming 97% of the primary structure in less than 2 h.
Bouchon
B
B
Transgène S.A., Strasbourg, France.
Jaquinod
M
M
Klarskov
K
K
Trottein
F
F
Klein
M
M
Van Dorsselaer
A
A
Bischoff
R
R
Roitsch
C
C
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D015801:Helminth Proteins; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry; D012550:Schistosoma mansoni / Q000378:metabolism*; D014357:Trypsin
D000947:Antigens, Helminth; D015801:Helminth Proteins; C052358:P28 antigen, Schistosoma; D010455:Peptides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D014357:Trypsin; D003488:Cyanogen Bromide