title: Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins.
abstract: 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 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.
  
  
  RESULTS
  The 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 1Da 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 α/β-ratio as screening parameters.
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Institute of Complex Molecular Systems, Eindhoven University of Technology, Expert Center Clinical Chemistry Eindhoven, The Netherlands; Laboratory for Clinical Chemistry and Hematology, Máxima Medisch Centrum, Veldhoven, Expert Center Clinical Chemistry Eindhoven, The Netherlands. Electronic address: florishelmich@gmail.com.
authors.forename: Floris
authors.initials: F
authors.lastname: Helmich
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Institute of Complex Molecular Systems, Eindhoven University of Technology, Expert Center Clinical Chemistry Eindhoven, The Netherlands.
authors.forename: Joost L J
authors.initials: JL
authors.lastname: van Dongen
authors.affiliation: Laboratory for Clinical Chemistry and Hematology, Máxima Medisch Centrum, Veldhoven, Expert Center Clinical Chemistry Eindhoven, The Netherlands.
authors.forename: Philip H M
authors.initials: PH
authors.lastname: Kuijper
authors.affiliation: Expert Center Clinical Chemistry Eindhoven, Clinical Laboratory, Catharina Hospital, Eindhoven, The Netherlands.
authors.forename: Volkher
authors.initials: V
authors.lastname: Scharnhorst
authors.affiliation: Laboratory of Chemical Biology, Department of Biomedical Engineering, Institute of Complex Molecular Systems, Eindhoven University of Technology, Expert Center Clinical Chemistry Eindhoven, The Netherlands.
authors.forename: Luc
authors.initials: L
authors.lastname: Brunsveld
authors.affiliation: Laboratory for Clinical Chemistry and Hematology, Máxima Medisch Centrum, Veldhoven, Expert Center Clinical Chemistry Eindhoven, The Netherlands.
authors.forename: Maarten A C
authors.initials: MA
authors.lastname: Broeren
mesh_terms: D000328:Adult; D000510:Alpha-Globulins / Q000032:analysis; D001609:Beta-Globulins / Q000032:analysis; D006453:Hemoglobinopathies / Q000175:diagnosis*; D006455:Hemoglobins, Abnormal / Q000032:analysis*; D006801:Humans; D008403:Mass Screening; D013058:Mass Spectrometry / Q000379:methods*; D012016:Reference Values; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013997:Time Factors
chemical_list: D000510:Alpha-Globulins; D001609:Beta-Globulins; D006455:Hemoglobins, Abnormal
keywords: Globulin; Hemoglobin; Hemoglobinopathy; Macromolecular; Mass spectrometry; Newborn screening; Q-ToF; Sickle cell; Thalassemia