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Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF
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Characterization of Structural Hemoglobin Variants by Top-Down Mass Spectrometry and R Programming Tools for Rapid

Yuan Lin1, Archana M Agarwal2,3, Alan G Marshall1,4

  • 1Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32308, United States.

Journal of the American Society for Mass Spectrometry
|December 27, 2021
PubMed

Hemoglobinopathies are one of the most prevalent genetic disorders, affecting millions throughout the world. These are caused by pathogenic variants in genes that control the production of hemoglobin (Hb) subunits. As the number of known Hb variants has increased, it has become more challenging to obtain unambiguous results from routine chromatographic assays employed in the clinical laboratory. Top-down proteomic analysis of Hb by mass spectrometry is a definitive method to directly characterize the sequences of intact subunits. Here, we apply "chimeric ion loading" to characterize Hb β subunit variants. In this technique, product ions derived from complementary dissociation techniques are accumulated in a multipole storage device before delivery to a 21 T Fourier-transform ion cyclotron resonance mass spectrometer for simultaneous detection. To further improve the efficiency of identification of Hb variants and localization of the mutation site(s), we developed an R programming script, "Variants Identifier", to search top-down data against a database containing accurate intact mass differences and diagnostic ions from investigated Hb variants. A second R script, "PredictDiag", was developed and employed to determine relevant diagnostic ions for additional Hb variants with known sequences. These two R scripts were successfully applied to the identification of a Hb δ-β fusion protein and other Hb variants. The combination of chimeric ion loading and the above R scripts enables rapid and reliable interpretation of top-down mass spectrometry data, regardless of activation type, for Hb variant identification.

Keywords:
FT-ICRFTMSFourier-transform ion cyclotron resonancechimeric ion loadingclinical mass spectrometrytop-down proteomics

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