Characterization of the glycation of albumin in freeze-dried and frozen human serum.
Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.
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National Institute of Standards and Technology, Galthersburg, Maryland 20899, USA.
D001800:Blood Specimen Collection; D015925:Cryopreservation; D019075:Electrophoresis, Capillary; D005612:Freeze Drying; D005615:Freezing; D017127:Glycation End Products, Advanced; D006031:Glycosylation; D006801:Humans; D010449:Peptide Mapping; D012016:Reference Values; D012709:Serum Albumin / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000093423:Glycated Serum Albumin
D017127:Glycation End Products, Advanced; D012709:Serum Albumin; D000093423:Glycated Serum Albumin