Characterization of Structural Hemoglobin Variants by Top-Down Mass Spectrometry and R Programming Tools for Rapid Identification

Abstract

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.

Surface sampling capillary electrophoresis–mass spectrometry for a direct chemical characterization of tissue and blood samples

“…However, the neurotransmitter GABA was only detected in tissue, as it is low‐abundant metabolite in blood at concentrations below 1 µM (Figure S8 ) [ 29 , 30 ]. Additionally, in contrast to tissue the major proteins peaks detected from dried blood are the α and β subunits of hemoglobin, which is expected to be the main protein in blood (Figure 3B ) [ 31 ]. Representative mass spectra are shown in Figure S9 to facilitate annotation.…”