title: Quantitative analysis of glycation sites on human serum albumin using (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: BACKGROUND
  One of the long term complications of diabetes is the non-enzymatic addition of glucose to proteins in blood, such as human serum albumin (HSA), which leads to the formation of an Amadori product and advanced glycation end products (AGEs). This study uses (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to provide quantitative data on the extent of modification that occurs in the presence of glucose at various regions in the structure of minimally glycated HSA.
  
  
  METHODS
  Normal HSA, with no significant levels of glycation, was digested by various proteolytic enzymes in the presence of water, while a similar sample containing in vitro glycated HSA was digested in (18)O-enriched water. These samples were then mixed and the (16)O/(18)O ratios were measured for peptides in each digest. The values obtained for the (16)O/(18)O ratios of the detected peptides for the mixed sample were used to determine the degree of modification that occurred in various regions of glycated HSA.
  
  
  RESULTS
  Peptides containing arginines 114, 81, or 218 and lysines 413, 432, 159, 212, or 323 were found to have (16)O/(18)O ratios greater than a cut off value of 2.0 (i.e., a cut off value based on results noted when using only normal HSA as a reference). A qualitative comparison of the (16)O- and (18)O-labeled digests indicated that lysines 525 and 439 also had significant degrees of modification. The modifications that occurred at these sites were variations of fructosyl-lysine and AGEs which included 1-alkyl-2-formyl-3,4-glycoyl-pyrole and pyrraline.
  
  
  CONCLUSIONS
  Peptides containing arginine 218 and lysines 212, 413, 432, and 439 contained high levels of modification and are also present near the major drug binding sites on HSA. This result is clinically relevant because it suggests the glycation of HSA may alter its ability to bind various drugs and small solutes in blood.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, NE, United States.
authors.forename: Omar S
authors.initials: OS
authors.lastname: Barnaby
authors.forename: Chunling
authors.initials: C
authors.lastname: Wa
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Cerny
authors.forename: William
authors.initials: W
authors.lastname: Clarke
authors.forename: David S
authors.initials: DS
authors.lastname: Hage
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017127:Glycation End Products, Advanced / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007553:Isotope Labeling; D010103:Oxygen Isotopes; D010446:Peptide Fragments / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D017127:Glycation End Products, Advanced; D010103:Oxygen Isotopes; D010446:Peptide Fragments; D004364:Pharmaceutical Preparations; D012709:Serum Albumin; D014357:Trypsin
references.citation: Diabetologia. 1995 Sep;38(9):1076-81
references.pmid: 8591822
references.citation: Biochem J. 1989 Aug 1;261(3):871-8
references.pmid: 2803249
references.citation: Clin Chim Acta. 2007 Oct;385(1-2):48-60
references.pmid: 17707360
references.citation: Rapid Commun Mass Spectrom. 2001;15(24):2456-65
references.pmid: 11746917
references.citation: Anal Chem. 2001 Jul 1;73(13):2836-42
references.pmid: 11467524
references.citation: Anal Biochem. 2009 Oct 1;393(1):8-22
references.pmid: 19524544
references.citation: Clin Chem. 1965 Apr;11:478-87
references.pmid: 14277286
references.citation: J Biol Chem. 1986 Oct 15;261(29):13542-5
references.pmid: 3759977
references.citation: J Mass Spectrom. 2005 Jul;40(7):969-72
references.pmid: 15768376
references.citation: Nature. 1992 Jul 16;358(6383):209-15
references.pmid: 1630489
references.citation: J Biol Chem. 2005 Feb 18;280(7):5724-32
references.pmid: 15557329
references.citation: Diabetes. 2003 Jul;52(7):1825-36
references.pmid: 12829653
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: Rapid Commun Mass Spectrom. 2000;14(14):1226-32
references.pmid: 10918372
references.citation: Proc Natl Acad Sci U S A. 1984 Jan;81(2):583-7
references.pmid: 6582514
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3316-9
references.pmid: 12824316
references.citation: J Pharmacol Exp Ther. 2006 May;317(2):667-75
references.pmid: 16410405
references.citation: J Am Soc Mass Spectrom. 2004 Apr;15(4):437-45
references.pmid: 15047049
references.citation: Mass Spectrom Rev. 2000 Sep-Oct;19(5):279-304
references.pmid: 11079246
references.citation: J Mol Biol. 2005 Oct 14;353(1):38-52
references.pmid: 16169013
references.citation: J Biol Chem. 1984 Mar 25;259(6):3812-7
references.pmid: 6706980
references.citation: Biochim Biophys Acta. 2003 Oct 13;1623(2-3):88-97
references.pmid: 14572906
references.citation: Biopharm Drug Dispos. 1997 Dec;18(9):791-801
references.pmid: 9429743
references.citation: Mass Spectrom Rev. 2006 Sep-Oct;25(5):775-97
references.pmid: 16625652
references.citation: Rapid Commun Mass Spectrom. 2005;19(19):2849-56
references.pmid: 16155976
references.citation: Anal Chem. 2006 Dec 1;78(23):7967-77
references.pmid: 17134129
references.citation: Bioinformatics. 2006 Nov 15;22(22):2739-45
references.pmid: 16954138
references.citation: Clin Chem. 1995 Nov;41(11):1654-61
references.pmid: 7586558
references.citation: J Mass Spectrom. 1997 Jul;32(7):773-8
references.pmid: 9241858
references.citation: Biochem J. 1999 Nov 15;344 Pt 1:109-16
references.pmid: 10548540
references.citation: Anal Biochem. 2006 Feb 15;349(2):229-41
references.pmid: 16356458
references.citation: Eur J Clin Pharmacol. 1993;45(6):577-9
references.pmid: 8157046
references.citation: J Agric Food Chem. 2003 Jan 1;51(1):51-7
references.pmid: 12502384
references.citation: Curr Med Chem. 2004 Aug;11(16):2185-93
references.pmid: 15279557
references.citation: Electrophoresis. 1997 Mar-Apr;18(3-4):403-8
references.pmid: 9150918
references.citation: J Chromatogr B Biomed Sci Appl. 2001 Aug 5;759(1):1-15
references.pmid: 11499613
references.citation: J Proteome Res. 2008 May;7(5):2025-32
references.pmid: 18396901
references.citation: Proteins. 2005 Dec 1;61(4):704-21
references.pmid: 16231289
references.citation: Biochem J. 2002 May 15;364(Pt 1):1-14
references.pmid: 11988070