Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA(1c) sample

Glycated hemoglobin (HbA(1c)) is a `gold standard' biomarker for assessing the glycemic index of an individual. HbA(1c) is formed due to nonenzymatic glycosylation at N-terminal valine residue of the P-globin chain. Cation exchange based high performance liquid chromatography (CE HPLC) is mostly used to quantify HbA(1c), 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 HbA(1c) (52.1%) in the CE HPLC method. The observed HbA(1c) 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 HbA(1c) and Hb Beckman might have resulted in the coelution of the variant with HbA(1c) in CE HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA(1c), it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA(1c) must be estimated using an alternative method. (C) 2015 Elsevier Inc. All rights reserved.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015