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Analysis of Minor Hemoglobins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

¹ Division of Hematology, and ² Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Zurich, Switzerland.

^aAddress correspondence to this author at: Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Fax 41-1-266-71-69; e-mail heinz.troxler{at}kispi.unizh.ch.

Background: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions.

Methods: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides.

Results: Analysis of the HbA₂ fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected - and -chains, the carbamylated - and the ß^S-chains. Additionally, we analyzed HbH, Hb Barts, HbA_1b, pre-HbA_1c, HbA_1c, HbF₁, HbF, HbA_1d3a, HbA_1d3b, HbA₂, and HbC₁ fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation.

Conclusions: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated - and the ß^S-chain elutes at the same time as normal HbA₂ (₂₂) and thus leads to falsely increased HbA₂ values in patients with HbS when blood is analyzed with PolyCAT A chromatography.

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				T. N. Higgins, A. Khajuria, and M. Mack Quantification of HbA2 in Patients With and Without {beta}-Thalassemia and in the Presence of HbS, HbC, HbE, and HbD Punjab Hemoglobin Variants: Comparison of Two Systems Am J Clin Pathol, March 1, 2009; 131(3): 357 - 362. [Abstract] [Full Text] [PDF]

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