Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA1c sample |
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Authors: | Rajdeep Das Monita Muralidharan Gopa Mitra Vijay Bhat Boby Mathew Debnath Pal Cecil Ross Amit Kumar Mandal |
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Affiliation: | 1. Clinical Proteomics Unit, Division of Molecular Medicine, St. John''s Research Institute, St. John''s National Academy of Health Sciences, Bangalore 560034, India;2. Manipal Hospital, Bangalore 560017, India;3. Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560012, India;4. Department of Medicine, St. John''s Medical College and Hospital, St. John''s National Academy of Health Sciences, Bangalore 560034, India |
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Abstract: | Glycated hemoglobin (HbA1c) is a ‘gold standard’ biomarker for assessing the glycemic index of an individual. HbA1c is formed due to nonenzymatic glycosylation at N-terminal valine residue of the β-globin chain. Cation exchange based high performance liquid chromatography (CE–HPLC) is mostly used to quantify HbA1c in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE–HPLC-based quantification, resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA1c (52.1%) in the CE–HPLC method. The observed HbA1c did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA1c and Hb Beckman might have resulted in the coelution of the variant with HbA1c in CE–HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA1c, it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA1c must be estimated using an alternative method. |
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Keywords: | Cation exchange HPLC HbA1c Hb Beckman Hemoglobin variant Mass spectrometry Glycemic index |
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