Characterization of pertussis toxin by LC–MS/MS

Liquid chromatography–mass spectrometry (LC–MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000...

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Bibliographic Details
Published in:Analytical biochemistry 2008-03, Vol.374 (1), p.16-24
Main Authors: Tummala, Manorama, Hu, Peifeng, Lee, Shwu-Maan, Robinson, Amy, Chess, Edward
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Chromatography, High Pressure Liquid - methods
Dual spray electrospray
Mass spectrometry
Methionine oxidation
Molecular Sequence Data
Molecular Weight
Oxidation-Reduction
Pertussis Toxin - chemistry
Pertussis Toxin - metabolism
Pertussis toxin protein sequence
Protein oxidation
Protein Subunits - chemistry
Spectrometry, Mass, Electrospray Ionization
Tandem Mass Spectrometry - methods
Trypsin - metabolism
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Summary:Liquid chromatography–mass spectrometry (LC–MS) with a dual spray electrospray ionization source has been used to measure the molecular weights of pertussis toxin (PT) subunits. Measurement accuracy better than 0.4 Da was achieved for all PT subunits in the molecular weight range of 11,000 to 27,000 Da. At this mass assignment accuracy level, the sequences of the PT subunits investigated in this study are easily determined based on molecular weight alone. The subunits 1, 2, and 5 of PT were observed to undergo oxidation under normal storage conditions as ammonium sulfate suspension at 2 to 8 °C. These oxidized subunits can be separated completely or partially by reverse-phase high-performance liquid chromatography (HPLC) from their native counterparts. For the determination of oxidation sites, the oxidized subunits and their nonoxidized counterparts were fraction collected, trypsin digested, and mapped by LC–MS. The oxidized peptides and their nonoxidized counterparts were further studied by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to confirm their identities. The methionines at position 212 of subunit 1, at position 89 of subunit 2, and at position 40 of subunit 5 were found to be the primary sites of oxidation.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2007.10.001