Donald Hunt (Presenter)
University of Virginia
Authorship: Elizabeth Duselis* (1), Jane Yang* (2), Scott Ugrin (1), Jeffrey Shabanowitz (1), Dave Herold (2), Donald Hunt (1)
(1) University of Virginia, (2) University of California San Diego
| Short Abstract Hemoglobin Charlottesville, a previously unreported hemoglobin variant, contains a S138X (X= I or L) substitution on the alpha subunit. The presence of the variant hemoglobin was detected during manual review of cation exchange HPLC and capillary electrophoresis data. The identity of the variant was assigned through LC-MS/MS analysis of the intact globin subunits, using novel techniques for intact protein interrogation: sequential ion/ion reactions and multiple fills of the ion/ion reaction products into the C-trap prior to Orbitrap analysis. The resulting sequence coverage of the alpha variant (141 residues, 15.1 kDa) was 80%. Coupled with HCD MS3 analysis of the C-terminal residues, 86.5% sequence coverage of the alpha variant was achieved. |
Long Abstract
The most abundant form of hemoglobin in adults is Hb A, a tetramer composed of two alpha subunits and two beta subunits. Single nucleotide polymorphisms in the encoding genes for the globin subunits result in variants that differ by a single amino acid. The presence of variant hemoglobin was detected during manual review of unresolved cation exchange HPLC and capillary electrophoresis data. Analysis by LC-MS revealed an alpha subunit variant that differed by 26 Da. For definitive identification, the primary structure of this variant was assessed through LC-MS/MS of the intact globin subunit, using novel techniques for intact protein characterization.
Analysis was performed on a modified Orbitrap Elite platform. The instrument is configured with a front-end reagent ion source to introduce reagent anions for both electron transfer dissociation (ETD) reactions and ion/ion proton transfer (IIPT) reactions. The variant subunit was fragmented with ETD reactions; the ETD product ions were subjected to charge-reducing IIPT reactions. The employment of the latter ion/ion reaction spreads the fragment ion current throughout the analytical range and concentrates the fragment ions into fewer charge states, which in turn eases spectral complexity and increases effective peak capacity. The concomitant loss in ion current due to neutralization and charge transfer from the ion/ion reactions can be compensated by filling the C-trap with multiple loads of the ETD/IIPT product ions prior to Orbitrap analysis. The signal-to-noise (S/N) enhancement of the fragment ion spectrum increases linearly with each C-trap fill; this is a distinct advantage over spectral averaging, where S/N scales with the square root of the number of spectra averaged. To extend sequence coverage into the C-terminal residues, the z15+2• ETD/IIPT product ion was selected and subjected to further fragmentation in the HCD cell. This MS3 analysis allowed assignment of a S138X (X= I or L) mutation of the alpha globin subunit; this previously unreported hemoglobin variant is now named Hemoglobin Charlottesville.
The intact variant subunit (141 residues, 15.1 kDa) was used for LC-MS/MS analysis. Utilization of ETD/IIPT sequential reactions and multiple fills of fragment ions into the C-trap resulted in 80% sequence coverage of the variant subunit. In combination with the HCD MS3, the sequence coverage totals 86.5%.
References & Acknowledgements:
Earley L; Anderson LC; Bai DL; Mullen C; Syka JEP; English AM; Dunyach JJ; Stafford GC; Shabanowitz J; Hunt DF; Compton PD. “Front-end electron transfer dissociation: a new ionization source.” Anal. Chem. 2013, 85, 8385-8390.
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