title: Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA(1c) sample.
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.
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Das
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India.
authors.forename: Monita
authors.initials: M
authors.lastname: Muralidharan
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India.
authors.forename: Gopa
authors.initials: G
authors.lastname: Mitra
authors.affiliation: Manipal Hospital, Bangalore 560017, India.
authors.forename: Vijay
authors.initials: V
authors.lastname: Bhat
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India.
authors.forename: Boby
authors.initials: B
authors.lastname: Mathew
authors.affiliation: Supercomputer Education and Research Centre, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Debnath
authors.initials: D
authors.lastname: Pal
authors.affiliation: Department of Medicine, St. John's Medical College and Hospital, St. John's National Academy of Health Sciences, Bangalore 560034, India.
authors.forename: Cecil
authors.initials: C
authors.lastname: Ross
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences, Bangalore 560034, India. Electronic address: amit@sjri.res.in.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Mandal
mesh_terms: D000595:Amino Acid Sequence; D001786:Blood Glucose / Q000032:analysis; D002411:Cation Exchange Resins; D002851:Chromatography, High Pressure Liquid; D003920:Diabetes Mellitus / Q000097:blood / Q000175:diagnosis; D003937:Diagnosis, Differential; D005189:False Positive Reactions; D005260:Female; D006442:Glycated Hemoglobin / Q000032:analysis; D006453:Hemoglobinopathies / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D006455:Hemoglobins, Abnormal / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008875:Middle Aged; D008970:Molecular Weight; D010449:Peptide Mapping; D017354:Point Mutation; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D013499:Surface Properties; D053719:Tandem Mass Spectrometry
chemical_list: D001786:Blood Glucose; D002411:Cation Exchange Resins; D006442:Glycated Hemoglobin; D006455:Hemoglobins, Abnormal; C517652:hemoglobin A1c protein, human; C000608608:hemoglobin Beckman
keywords: Cation exchange HPLC; Glycemic index; Hb Beckman; HbA(1c); Hemoglobin variant; Mass spectrometry