Background: Screening for the identification of abnormal hemoglobins is of great clinical importance, especially in populations where the prevalence of hemoglobinopathies is not negligible. The definitive characterization of the hemoglobin variant takes place by DNA sequencing, but this investigation does not take into account possible protein post-translational modifications. Methods: Routine first-level Hb analysis was performed by hemolysate CE-HPLC separation. The hemolysate was also subjected to MALDI-TOF analysis using CHCA matrix and to MALDI-TOF ISD using 1.5-DAN matrix in both linear and reflector mode. To gain insight on the a, b, and y ions, the single ISD fragment of interest underwent PSD analysis. β globin gene exon sequencing was carried out by Sanger method. Results: The CE-HPLC analysis of the hemolysate revealed a peak with a retention time superimposable to HbA1c with approximately 50% relative abundance. To characterize the anomalous Hb peak, a preliminary MALDI-TOF analysis of the hemolysate was performed, revealing a double peak at the beta chains level with a delta of +14 m/z. Then, MALDI-ISD combined with PSD analyses uniquely established the N-terminal sequence of the first eight aminoacid AHLTPEEK, corresponding to Hb Raleigh with the N-acetylated aminoacidic end. DNA analysis confirmed the GTG→GCG substitution at codon one of beta exon 1 corresponding to a V→A. Conclusions: Our results showed that the MALDI-ISD MS analysis of the whole hemolysate is a rapid and effective method for Hb characterization and, above all, it may allow easily the identification of post-translational modifications that are not detectable through DNA analysis.

MALDI-ISD mass spectrometry analysis as a simple and reliable tool to detect post-translational modifications of hemoglobin variants: the case of Hb Raleigh

Selene De Benedittis;Patrizia Spadafora;Olivier Gallo;Gemma Di Palma;Francesca Cavalcanti;Luigi Citrigno;Antonio Qualtieri
2023

Abstract

Background: Screening for the identification of abnormal hemoglobins is of great clinical importance, especially in populations where the prevalence of hemoglobinopathies is not negligible. The definitive characterization of the hemoglobin variant takes place by DNA sequencing, but this investigation does not take into account possible protein post-translational modifications. Methods: Routine first-level Hb analysis was performed by hemolysate CE-HPLC separation. The hemolysate was also subjected to MALDI-TOF analysis using CHCA matrix and to MALDI-TOF ISD using 1.5-DAN matrix in both linear and reflector mode. To gain insight on the a, b, and y ions, the single ISD fragment of interest underwent PSD analysis. β globin gene exon sequencing was carried out by Sanger method. Results: The CE-HPLC analysis of the hemolysate revealed a peak with a retention time superimposable to HbA1c with approximately 50% relative abundance. To characterize the anomalous Hb peak, a preliminary MALDI-TOF analysis of the hemolysate was performed, revealing a double peak at the beta chains level with a delta of +14 m/z. Then, MALDI-ISD combined with PSD analyses uniquely established the N-terminal sequence of the first eight aminoacid AHLTPEEK, corresponding to Hb Raleigh with the N-acetylated aminoacidic end. DNA analysis confirmed the GTG→GCG substitution at codon one of beta exon 1 corresponding to a V→A. Conclusions: Our results showed that the MALDI-ISD MS analysis of the whole hemolysate is a rapid and effective method for Hb characterization and, above all, it may allow easily the identification of post-translational modifications that are not detectable through DNA analysis.
2023
Istituto per la Ricerca e l'Innovazione Biomedica -IRIB
hemoglobin variant; in-source decay; mass spectrometry; post-translational modification
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14243/461503
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