Jane Yang (Presenter)
UCSD CALM
Authorship: Jane Y. Yang* (1), Elizabeth Dueslis* (2), Scott Ugrin (2), Jeffrey Shabanowitz (2), Donald Hunt (2), David A. Herold (1)
(1) University of California San Diego, (2) University of Virginia, *These authors contributed equally to this work.
| Short Abstract Hemoglobin Charlottesville, a previously unreported hemoglobin variant, contains a S138I/L substitution. The presence of the variant hemoglobin was detected during manual review of cation exchange HPLC and capillary electrophoresis data. From our dilute and shoot LC-MS approach, we predicted an amino acid mass difference of 26 in the alpha subunit with polarity of the residue going from polar to non-polar. ETD fragmentation confirmed the predictions and narrowed down the substitution position to 131, 133, or 138. Subsequent MS3 by HCD revealed that the substitution occurred at position 138 of the alpha subunit. |
Long Abstract
Hemoglobin variants due to a single nucleotide polymorphism typically contain single amino acid substitution. This substitution can be predicted based on the mass difference between normal subunit mass and the variant subunit mass.
First we identified the presence of the variant during manual review of cation exchange HPLC and capillary electrophoresis data, an unresolved peak to the right of A2 on the chromatogram and to the left of A2 on the electrophoretogram. Then we used a dilute and shoot LC-MS approach to determine information about the variant – mass difference of 26 on the alpha subunit with amino acid residue polarity going from polar to non-polar based on chromatographic elution time. Based on constraints of DNA base substitutions, the amino acid mass difference of 26 is due to one of four substitutions, His >Tyr, Ala>Pro, Ser>Leu, and Ser>Ile. Hence, we predicted the Ser>Ile/Leu substitution due to the change in polarity of the alpha chain of the hemoglobin. This was confirmed by LC-MS/MS with ETD fragmentation, which also narrowed down the location of the substitution at position 131, 133, or 138. Subsequent MS3 via HCD resulted in the assignment of the Ser138Ile/Leu substitution, a previously unreported hemoglobin variant, Hemoglobin Charlottesville.
The authors acknowledge NIH GM 037537 to DFH for support.
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