Identification of protein succination as a novel modification of tubulin.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and ß-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 µM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12ß and Cys303ß as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin-protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αß-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics.

Equivalence of protein inventories obtained from formalin-fixed paraffin-embedded and frozen tissue in multidimensional liquid chromatography-tandem mass spectrometry shotgun proteomic analysis.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 microg of protein yielded approximately 400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from approximately 17% after one year of storage to approximately 25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.

Improved methods for the enrichment and analysis of glycated peptides.

Nonenzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. Herein we report improved methods for the enrichment and analysis of glycated peptides using boronate affinity chromatography and electron-transfer dissociation mass spectrometry, respectively. The enrichment of glycated peptides was improved by replacing an off-line desalting step with an online wash of column-bound glycated peptides using 50 mM ammonium acetate, followed by elution with 100 mM acetic acid. The analysis of glycated peptides by MS/MS was improved by considering only higher charged (> or = 3) precursor ions during data-dependent acquisition, which increased the number of glycated peptide identifications. Similarly, the use of supplemental collisional activation after electron transfer (ETcaD) resulted in more glycated peptide identifications when the MS survey scan was acquired with enhanced resolution. Acquiring ETD-MS/MS data at a normal MS survey scan rate, in conjunction with the rejection of both 1+ and 2+ precursor ions, increased the number of identified glycated peptides relative to ETcaD or the enhanced MS survey scan rate. Finally, an evaluation of trypsin, Arg-C, and Lys-C showed that tryptic digestion of glycated proteins was comparable to digestion with Lys-C and that both were better than Arg-C in terms of the number of glycated peptides and corresponding glycated proteins identified by LC-MS/MS.

Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry.

Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.

Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products.

Immunohistochemical approaches have been widely used in the localization and quantification of advanced glycation end-products (AGEs). Traditional approaches for production of anti-AGE antibodies use cross-linkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to conjugate the AGE antigen to the carrier protein. However, these approaches often fail to produce antibodies that are specific to the particular AGE of interest. In the present study, Nepsilon-(carboxymethyl)lysine (CML), a major antigenic AGE structure, was conjugated to human serum albumin (HSA) using various cross-linkers, including EDC, bis(sulfosuccinimidyl)suberate (BS3) and glutaraldehyde, to compare their efficiency for the production of epitope-specific antibodies. All of the cross-linkers tested were capable of conjugating CML to HSA, and each CML-conjugated HSA was recognized by previously characterized anti-CML antibody. However, only the use of glutaraldehyde as the cross-linker resulted in the production of a CML-specific monoclonal antibody, termed 2G11. 2G11 significantly recognized CML-modified HSA and peptide, whereas it did not recognize Nepsilon-(carboxyethyl)lysine (CEL)-modified HSA and peptide, indicating that 2G11 is highly specific to CML, and can distinguish the difference of a single methyl group between the two epitopes. To further demonstrate the use of glutaraldehyde, anti-AGE antibodies against CEL, S-(2-succinyl)cysteine and S-(carboxymethyl)cysteine were obtained by conjugation with glutaraldehyde. These studies demonstrate the efficacy of glutaraldehyde as a cross-linker for the production of antibodies against small molecules.

Increased methionine sulfoxide content of apoA-I in type 1 diabetes.

Cardiovascular disease is a major cause of morbidity and premature mortality in diabetes. HDL plays an important role in limiting vascular damage by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells. Methionine (Met) residues in apolipoprotein A-I (apoA-I), the major apolipoprotein of HDL, reduce peroxides in HDL lipids, forming methionine sulfoxide [Met(O)]. We examined the extent and sites of Met(O) formation in apoA-I of HDL isolated from plasma of healthy control and type 1 diabetic subjects to assess apoA-I exposure to lipid peroxides and the status of oxidative stress in the vascular compartment in diabetes. Three tryptic peptides of apoA-I contain Met residues: Q(84)-M(86)-K(88), W(108)-M(112)-R(116), and L(144)-M(148)-R(149). These peptides and their Met(O) analogs were identified and quantified by mass spectrometry. Relative to controls, Met(O) formation was significantly increased at all three locations (Met(86), Met(112), and Met(148)) in diabetic patients. The increase in Met(O) in the diabetic group did not correlate with other biomarkers of oxidative stress, such as N(epsilon)-malondialdehyde-lysine or N(epsilon)-(carboxymethyl)lysine, in plasma or lipoproteins. The higher Met(O) content in apoA-I from diabetic patients is consistent with increased levels of lipid peroxidation products in plasma in diabetes. Using the methods developed here, future studies can address the relationship between Met(O) in apoA-I and the risk, development, or progression of the vascular complications of diabetes.

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry.

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.

Formation of methionine sulfoxide during glycoxidation and lipoxidation of ribonuclease A.

Chemical modification of proteins by reactive oxygen species affects protein structure, function and turnover during aging and chronic disease. Some of this damage is direct, for example by oxidation of amino acids in protein by peroxide or other reactive oxygen species, but autoxidation of ambient carbohydrates and lipids amplifies both the oxidative and chemical damage to protein and leads to formation of advanced glycoxidation and lipoxidation end-products (AGE/ALEs). In previous work, we have observed the oxidation of methionine during glycoxidation and lipoxidation reactions, and in the present work we set out to determine if methionine sulfoxide (MetSO) in protein was a more sensitive indicator of glycoxidative and lipoxidative damage than AGE/ALEs. We also investigated the sites of methionine oxidation in a model protein, ribonuclease A (RNase), in order to determine whether analysis of the site specificity of methionine oxidation in proteins could be used to indicate the source of the oxidative damage, i.e. carbohydrate or lipid. We describe here the development of an LC/MS/MS for quantification of methionine oxidation at specific sites in RNase during glycoxidation or lipoxidation by glucose or arachidonate, respectively. Glycoxidized and lipoxidized RNase were analyzed by tryptic digestion, followed by reversed phase HPLC and mass spectrometric analysis to quantify methionine and methionine sulfoxide containing peptides. We observed that: (1) compared to AGE/ALEs, methionine sulfoxide was a more sensitive biomarker of glycoxidative or lipoxidative damage to proteins; (2) regardless of oxidizable substrate, the relative rate of oxidation of methionine residues in RNase was Met29>Met30>Met13, with Met79 being resistant to oxidation; and (3) arachidonate produced a significantly greater yield of MetSO, compared to glucose. The methods developed here should be useful for assessing a protein's overall exposure to oxidative stress from a variety of sources in vivo.

Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis.

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.

Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease.

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

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.