The study of hemoglobin (Hb) disorders originated more than a 100 years ago with the discovery of sickle cell disease. The growing understanding of Hb has driven further studies eventually encompassing disorders associated with expression (thalassemias) and structural variation (hemoglobinopathies) of the protein. Currently, there are over 1000 known Hb variants. While the majority of severe clinical symptoms can be attributed to only a few Hb variants, their high prevalence in certain populations necessitates screening. Screening for Hb variants typically includes HPLC (high performance liquid chromatography) and CZE (capillary zone electrophoresis).(Kohne, 2011) Although valuable, these methods cannot definitively identify all clinically important Hb variants. Thus, various secondary tests are also required for definitive identification of Hb variants and can include expensive targeted molecular techniques, which may still miss an underlying condition if chosen inappropriately.
Mass spectrometry (MS) attempts to revisit the classic problem of Hb variant identification by applying sensitive, specific, and orthogonal analyte characterization via differences in mass due to amino acid substitutions or deletions.(Théberge, Infusini, Tong, McComb, & Costello, 2011) MS is capable of detecting individual Hb subunits, and providing accurate masses and relative abundances for alpha, beta or gamma subunits. These strengths of MS testing may help determine gene mutation location (i.e. alpha or beta) and direct further molecular testing. MS can also provide expressed Hb sequence information by possibly identifying mutation sites. Examples of Hb subunits identified through this approach included alpha, beta, and gamma (G and A) as well as several subunit variants such as sickle cell (beta) and G-Philadelphia (alpha). Identification of G-Philadelphia provides critical diagnostic information, as both Hb D and G-Philadelphia are indistinguishable during conventional screening.
The implementation of MS-based Hb methods is technically and analytically demanding. A typical mass spectrum may have hundreds of ions displayed by their mass-to-charge (m/z) ratios. These data are not easily interpreted. There is a significant need for better, more streamlined software solutions to produce straightforward result summary. Furthermore, resolution requirements in MS methods are far beyond the capabilities of currently available MS instruments in the clinical setting (TOF and Orbitrap mass analyzers). Due to overlapping spectral complexity, MS instruments need to detect 0.01 Da mass difference to distinguish Hb D from Hb A as an example. Fragmentation and/or digestion strategies can alleviate this problem by reducing the mass of the ions, thus reducing the resolving power needed. Strategies for fragmentation include top-down, middle-down or bottom-up approaches. The top-down approach ionizes the whole protein and performs fragmentation in the mass spectrometer. The benefit of this approach is minimal sample preparation, despite being software demanding (correlating fragments to original sequence) and potentially more variable. The bottom-up approach requires protein digestion, and the measurement is accomplished on the peptide level. This technique is less variable and software demanding, but requires much more sample preparation before MS/MS analysis leading to higher testing cost.(Rifai, Horvath, & Wittwer, Ch. 21) A compromise can be achieved by implementing middle-down sample preparation in which a less arduous digestion creates larger oligopeptides than typical bottom-up approaches. This technique can provide the resolution necessary, but without a demanding sample preparation.
The addition of MS to traditional Hb workflows has potential to provide critical diagnostic information to reach accurate clinical conclusions and diagnoses. Before wider adoption of the technique, efforts need to be made to make the platform more clinical lab-friendly, such as simpler sample preparation procedures and more robust data interpretation. The latter is highly dependent on software that can accurately translate the raw m/z data to the hemoglobinopathy or likely thalassemia with little guidance from the pathologist.
Kohne, E. (2011). Hemoglobinopathies: Clinical Manifestations, Diagnosis, and Treatment. Deutsches Ärzteblatt International, 108(31-32), 532-540. doi: 10.3238/arztebl.2011.0532
Rifai, N., Horvath, A. R., & Wittwer, C. Tietz textbook of clinical chemistry and molecular diagnostics (Sixth edition. ed., pp. 327-327.e21). Retrieved from https://www.clinicalkey.com/dura/browse/bookChapter/3-s2.0-C20140016450.
Théberge, R., Infusini, G., Tong, W., McComb, M. E., & Costello, C. E. (2011). Top-Down Analysis of Small Plasma Proteins Using an LTQ-Orbitrap. Potential for Mass Spectrometry-Based Clinical Assays for Transthyretin and Hemoglobin. International journal of mass spectrometry, 300(2-3), 130-142. doi: 10.1016/j.ijms.2010.08.012