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

• A new approach for hemoglobin screening is proposed by top-down mass spectrometry.
• Screening parameters comprise the α- and β-globulin mass and their intensity ratio.
• Pre-analysis is reduced to cell lysis only and acquisition takes one minute.
• In a clinical study, all patients with hemoglobin variants were resolved.
• Hb-variants with 1 Da mass differences are resolved, even in heterozygous state.

BackgroundGiven 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.MethodsBy 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 α- and β-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/β-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared.ResultsThe α/β-ratio is a poor marker for thalassemia patients, yet deviant α/β-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.ConclusionsAlthough 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 α/β-ratio as screening parameters.