Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins

Background: Given the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions. Methods: By parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of alpha- and beta-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (alpha/beta-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared. Results: The alpha/beta-ratio is a poor marker for thalassemia patients, yet deviant alpha/beta-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1 Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval. Conclusions: Although subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the alpha/beta-ratio as screening parameters. (C) 2016 Elsevier B.V. All rights reserved.