title: Complete amino acid sequencing and immunoaffinity clean-up can facilitate screening of various chemical modifications on human serum albumin.
abstract: This manuscript describes a simple and practical strategy for screening of various chemical modifications of human serum albumin (HSA). Serum albumin is the most abundant blood plasma protein in humans (HSA, 66.5 kDa, t1/2 = 19 d), constituting about 60 % of total proteins. Therefore, it is believed to be the main target of chemical stresses during physiological events such as increased oxidative stress from the degenerative diseases of aging, and higher glucose stress in diabetes mellitus. Consequently, chemical modifications can provide significant information about these biological events. In this study, a complete and robust sequencing method was attained by the peptide mass fingerprinting (PMF) technique using two different complementary proteases (trypsin and Glu-C) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) in both positive and negative ionization modes. Using this strategy, several modified peptides, 12 oxidations, 25 glycations, 6 lipoxidations, and 5 nitrations have been identified on HSA treated with chemical reactions in vitro. Combined with immunoaffinity clean-up, this method was able to detect in vivo chemical modifications of HSA and found oxidized Trp(214) and glycated Lys(525) in healthy human plasma.
authors.affiliation: Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, 980-8578, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Goto
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Murata
authors.forename: Seon Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Oe
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010455:Peptides / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013312:Stress, Physiological; D014357:Trypsin / Q000737:chemistry
chemical_list: D010455:Peptides; D012709:Serum Albumin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin