Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease.
Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N(epsilon)-(carboxymethyl)lysine (CML). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degrees C for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CML formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CML is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
Jonathan W C
JW
Brock
Davinia J S
DJ
Hinton
William E
WE
Cotham
Thomas O
TO
Metz
Suzanne R
SR
Thorpe
John W
JW
Baynes
Jennifer M
JM
Ames
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002853:Chromatography, Liquid; D005947:Glucose / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D008024:Ligands; D008239:Lysine / Q000031:analogs & derivatives / Q000096:biosynthesis*; D013058:Mass Spectrometry; D008745:Methylation; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012260:Ribonucleases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D000596:Amino Acids; D017127:Glycation End Products, Advanced; D008024:Ligands; D011506:Proteins; D012260:Ribonucleases; D005947:Glucose; D008239:Lysine