Analyses using different MS-based methods were carried out in a three-month clinical trial. 2017 clinical samples were analysed to determine HbA_2 levels, screen for clinically significant variants and identify additional rare variants. HbA_2 level determination showed good average correlation to HPLC values using both intact globin chain analysis and MRM targeted peptide analysis. Mass error plots from intact protein analysis gave an indication of the presence of α- and β-chain variants which could then be investigated and characterised. Hemoglobin variants not identified using current screening methods were able to be characterised (i.e. Hb Tyne, HbJ-Baltimore and HbJ-Broussais). The MRM-based method confirmed the presence of HbS, HbC, HbE, and HbO-Arab. The method can potentially be modified to monitor for the presence of other α- and β-chain variants such as HbG Philadelphia which was later added to our method. Further optimisation for screening of the HbD-Punjab mutation is necessary. Top-down ETD method can identify -1 Da variants without the lengthy enzymatic digestion sample preparation step. It provided reliable confirmation of HbS and HbC mutations and indication of HbE, HbO-Arab and HbD-Punjab mutations. All three methods show promise for diagnostic application providing definitive diagnosis and confirmation of the presence of clinically significant variants. All MS-based screening methods have been semi-automated and can potentially be fully automated in future to be introduced for clinical use in a screening hospital.
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