MALDI-ISD Mass Spectrometry Analysis of Hemoglobin Variants: a Top-Down Approach to the Characterization of Hemoglobinopathies

• Published in: Journal of the American Society for Mass Spectrometry 2015-08, Vol.26 (8), p.1299-1310
• Main Authors: Théberge, Roger, Dikler, Sergei, Heckendorf, Christian, Chui, David H. K., Costello, Catherine E., McComb, Mark E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analytical Chemistry; Bioinformatics; Biotechnology; Chains; Chemistry; Chemistry and Materials Science; Desorption; Diagnostic systems; Fragmentation; Fragments; Hemoglobin; Hemoglobinopathies - blood; Hemoglobins - analysis; Hemoglobins - chemistry; Humans; Ionization; Ions; Lasers; Mass spectrometry; Molecular Sequence Data; Organic Chemistry; Peptide Mapping - methods; Proteomics; Research Article; Scientific imaging; Screening; Sequence Analysis, Protein - methods; Sequences; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Spectroscopy
• ISSN: 1044-0305; 1879-1123; 1879-1123
• DOI: 10.1007/s13361-015-1164-4

Hemoglobinopathies are the most common inherited disorders in humans and are thus the target of screening programs worldwide. Over the past decade, mass spectrometry (MS) has gained a more important role as a clinical means to diagnose variants, and a number of approaches have been proposed for characterization. Here we investigate the use of matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) with sequencing using in-source decay (MALDI-ISD) for the characterization of Hb variants. We explored the effect of matrix selection using super DHB or 1,5-diaminonaphthalene on ISD fragment ion yield and distribution. MALDI-ISD MS of whole blood using super DHB simultaneously provided molecular weights for the alpha and beta chains, as well as extensive fragmentation in the form of sequence defining c-, (z + 2)-, and y-ion series. We observed sequence coverage on the first 70 amino acids positions from the N- and C-termini of the alpha and beta chains in a single experiment. An abundant beta chain N-terminal fragment ion corresponding to βc34 was determined to be a diagnostic marker ion for Hb S (β6 Glu→Val, sickle cell), Hb C (β6 Glu→Lys), and potentially for Hb E (β26 Glu→Lys). The MALDI-ISD analysis of Hb S and HbSC yielded mass shifts corresponding to the variants, demonstrating the potential for high-throughput screening. Characterization of an alpha chain variant, Hb Westmead (α122 His→Gln), generated fragments that established the location of the variant. This study is the first clinical application of MALDI-ISD MS for the determination and characterization of hemoglobin variants. Graphical Abstract ᅟ