title: MALDI-ISD Mass Spectrometry Analysis of Hemoglobin Variants: a Top-Down Approach to the Characterization of Hemoglobinopathies.
abstract: 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.
authors.affiliation: Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, MA, 02118, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Théberge
authors.forename: Sergei
authors.initials: S
authors.lastname: Dikler
authors.forename: Christian
authors.initials: C
authors.lastname: Heckendorf
authors.forename: David H K
authors.initials: DH
authors.lastname: Chui
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Costello
authors.forename: Mark E
authors.initials: ME
authors.lastname: McComb
mesh_terms: D000595:Amino Acid Sequence; D006453:Hemoglobinopathies / Q000097:blood; D006454:Hemoglobins / Q000032:analysis* / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D006454:Hemoglobins