Received on January 7, 2005
Accepted on March 14, 2005

Hematology

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

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

^* To whom correspondence should be addressed. 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 ^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 ^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.