Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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 alpha- and delta-chains, the carbamylated alpha- and the betaS-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 alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.
Zurbriggen
Karin
K
Division of Hematology, University of Zurich, Zurich, Switzerland.
Schmugge
Markus
M
Schmid
Marlis
M
Durka
Silke
S
Kleinert
Peter
P
Kuster
Thomas
T
Heizmann
Claus W
CW
Troxler
Heinz
H
D002851:Chromatography, High Pressure Liquid; D005188:False Negative Reactions; D005319:Fetal Hemoglobin / Q000032:analysis / Q000378:metabolism; D006442:Glycated Hemoglobin / Q000032:analysis / Q000378:metabolism; D006443:Hemoglobin A2 / Q000032:analysis / Q000378:metabolism; D006444:Hemoglobin C / Q000032:analysis / Q000378:metabolism; D006447:Hemoglobin H / Q000032:analysis / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000032:analysis / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis* / Q000378:metabolism; D006455:Hemoglobins, Abnormal / Q000032:analysis / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017086:beta-Thalassemia / Q000097:blood
D005319:Fetal Hemoglobin; D006442:Glycated Hemoglobin; D006443:Hemoglobin A2; D006444:Hemoglobin C; D006447:Hemoglobin H; D006451:Hemoglobin, Sickle; D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; C030448:hemoglobin Bart's