Proteomic Analysis of the Site Specificity of Glycation and Carboxymethylation of Ribonuclease

Abstract

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.

Proteomic Analysis of Arginine Adducts on Glyoxal-modified Ribonuclease

“…The recovered protein was reduced with DTT, derivatized with 4-vinylpyridine, and digested with trypsin (enzyme:substrate ratio of 5:100 (w/w)) at 37°C for 5 h. All samples were prepared in triplicate. The procedures have been published in detail elsewhere (11).…”

“…Control incubations used phosphate buffer in place of HA solution. Further aliquots of the 7-day RNase-1 mM glyoxal incubation (40 l) were ultrafiltrated to remove any unreacted glyoxal (11), resuspended in an equivalent volume of 0.4 M, pH 7.4 phosphate buffer (40 l) containing DTPA (final concentration 0.1 mM) and incubated for 1 and 7 days.…”

“…Tryptic Digestion of HA Incubations-Incubations were digested using a modification of the procedure reported by Brock et al (11). Protein (0.5 mg) was diluted into 50 l of 0.1 M MOPS buffer containing 6 M urea and 1 mM EDTA.…”

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A

“…Tryptic digestion of RNase was performed as described previously [8]. Briefly, RNase (1 mg) was suspended in 75 μl of MOPS buffer (100 mM, pH 7.2) containing urea (6 M) and EDTA (1 mM).…”

“…Quantification of peptides was performed with the Quattro set in full scan mode (200 -1800 amu), and masses of interest were extracted using MassLynx (Micromass) software. Fractional modification of Met-containing peptides was calculated as described previously [8]. Briefly, relative amounts of unmodified peptides were determined by summing the peak areas of all charged forms of the peptide of interest and dividing by the sum of the peak areas of the charged forms of an internal reference peptide in RNase, H 105 -V 124 .…”