Profiling of Methylglyoxal Blood Metabolism and ... - ACS Publications

Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Profiling of Methylglyoxal Blood Metabolism and Advanced Glycation End-Product Proteome Using a Chemical Probe Christian Sibbersen,†,§,∥,⊥ Anne-Mette Schou Oxvig,†,§,∥,⊥ Sarah Bisgaard Olesen,†,§,∥ Camilla Bak Nielsen,† James J. Galligan,‡ Karl Anker Jørgensen,§ Johan Palmfeldt,∥ and Mogens Johannsen*,† †

Department of Forensic Medicine, Aarhus University, Aarhus 8200, Denmark Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721, United States § Department of Chemistry, Aarhus University, Aarhus 8000, Denmark ∥ Department of Clinical Medicine, Aarhus University Hospital, Aarhus 8000, Denmark

ACS Chem. Biol. Downloaded from pubs.acs.org by UNIV OF RHODE ISLAND on 12/04/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: Methylglyoxal (MG) is quantitatively the most important precursor to advanced glycation end-products (AGEs), and evidence is accumulating that it is also a causally linked to diabetes and aging related diseases. Living systems primarily reside on the glyoxalase system to detoxify MG into benign D-lactate. The flux to either glycation or detoxification, accordingly, is a key parameter for how well a system handles the ubiquitous glyoxal burden. Furthermore, insight into proteins and in particular their individual modification sites are central to understanding the involvement of MG and AGE in diabetes and aging related diseases. Here, we present a simple method to simultaneously monitor the flux of MG both to D-lactate and to protein AGE formation in a biological sample by employing an alkyne-labeled methylglyoxal probe. We apply the method to blood and plasma to demonstrate the impact of blood cell glyoxalase activity on plasma protein AGE formation. We move on to isolate proteins modified by the MG probe and accordingly can present the first general inventory of more than 100 proteins and 300 binding sites of the methylglyoxal probe on plasma as well as erythrocytic proteins. Some of the data could be validated against a number of in vivo and in vitro targets for advanced glycation previously known from the literature; the majority of proteins and specific sites however were previously unknown and may guide future research into MG and AGE to elucidate how these are functionally linked to diabetic disease and aging.

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quantitatively the most important precursor to AGE generation.9 Knowledge about the proportion of MG metabolized versus MG forming AGEs is accordingly of high importance to understand disease progression. In the case of diabetes, the degree of protein modification is directly correlated with the cellular concentration of MG, which is mainly controlled by the glycolytic flux as well as the activity of the glyoxalase system and levels of glutathione.4,10,11 Another equally important aspect of MG function is to determine which proteins are susceptible to AGE modification. Though AGEs on a general scalehave been strongly associated with late diabetic complications and aging related diseases, the question of which specific proteins are post-translationally modified remains largely unknown.9 Identification of proteins that are prone to modification is essential to beginning to elucidate the

n recent years, it has become apparent that a growing number of endogenous small molecules play alternative biological roles to their canonical function. In addition to being, e.g., an energy source or metabolite, these compounds also regulate the activity of enzymes by forming posttranslational modifications and, in some cases, noncovalent interactions.1−4 One such metabolite is methylglyoxal (MG), a toxic byproduct of the glycolytic pathway. MG is primarily metabolized via glyoxalase I and II (GLO1 and 2) into Dlactate.5 Due to the relative abundance of MG (∼0.1−0.4% of the total glycolytic flux), a fraction escapes detoxification and, based on its electrophilic nature, generates advanced glycation end-products (AGEs) via nonenzymatic reaction with nucleophilic amino acid side chains. The most prominent AGEs are the arginine-derived hydroimidazolone MG-H1 and the less abundant lysine derived Nε-(1-carboxyethyl)lysine (CEL).6 AGEs are a class of protein post-translational modifications that have been strongly associated with late diabetic complications and biological aging.7,8 Although the majority of MG (>99%) is converted to D-lactate, MG is still © XXXX American Chemical Society

Received: August 7, 2018 Accepted: November 13, 2018

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DOI: 10.1021/acschembio.8b00732 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Figure 1. Metabolism and protein reactivity of the methylglyoxal probe alkMG in human whole blood. Akin to MG, alkMG is primarily metabolized intracellularly and is exported from the erythrocytes as its lactate analogue, alkLactate. A fraction, however, escapes detoxification, resulting in post-translational modifications of both plasma and erythrocytic proteins.

Figure 2. alkMG metabolized into alkLactate in whole blood and enriched to improve detection compared to endogenous lactate from methylglyoxal. (A) Whole blood was incubated with or without 5 mM MG for 2 h at 37 °C and, following removal of blood cells and plasma proteins, analyzed by untargeted LC-QTOF-MS. The m/z value of lactate (green) was extracted from the base peak chromatogram. (B) Whole blood was incubated with or without 5 mM MG probe (alkMG) for 2 h. Following removal of blood cells and plasma proteins, metabolites of alkMG were enriched on streptavidin resin and released prior to LC-QTOF-MS analysis. The m/z values of the unreacted azide linker (blue; calculated m/z = 179.0928, measured = 179.0936) and the lactate derivative (red; calculated m/z = 307.1401, measured = 307.1400) were extracted in the chromatograms. B


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Figure 3. Workflow applied to study alkMG metabolism and protein modification in whole blood, plasma, or erythrocyte lysate. In brief, after incubation with alkMG, blood cells are removed by centrifugation and remaining plasma proteins separated from small molecules by precipitation. The plasma supernatant containing the metabolites is conjugated to a cleavable azido-azo-biotin linker (Figure S6) using a click-chemistry-based protocol followed by enrichment on a streptavidin−agarose resin.19 The labeled metabolites are released by reduction of the azobenzene moiety and analyzed by LC-QTOF-MS. Similarly, the pellet of plasma proteins is dissolved and trypsinized, and binding-site peptides are enriched prior to nanoLC−MS/MS analysis. Aliquots of labeled proteins are furthermore reacted with rhodamine-azide to visualize protein binding by fluorescence. A similar workflow was applied for experiments using isolated plasma or erythrocyte lysate.

showing the most intense ion at any time, the formation of lactate is not directly evident (Figure 2A). However, through the extraction of the molecular ion of lactate, it is clear that lactate levels increase upon the addition of MG when compared to the control, indicating active glyoxalase metabolism in the blood sample. To validate alkMG as a serviceable surrogate for MG, we next performed the same analysis using 5 mM alkMG. Following incubation and precipitation of cells and proteins, the soluble alkMG derived plasma metabolites from the supernatant were enriched using our previously developed cleavable azidonylated resin (workflow similar to Figure 3 top).16 Following dithionite cleavage, supernatants were analyzed via LC-QTOF-MS, similar to the MG sample (Figure 2B). In parallel, we chemically synthesized and analyzed an aqueous reference sample containing alkLactate, the expected small metabolite product from glyoxalase-catalyzed metabolism of alkMG, in order to verify possible product formation (see Figure S1 in SI). The most intense peak in the base-peak chromatogram not present in a blank control corresponded to the peak found in the alkLactate reference sample. Furthermore, the signal-to-noise (s/n) ratio of alkLactate was approximately 1000, which is 100-fold higher than that seen in the samples using native MG (Figure 2A). To validate that alkMG is metabolized by the glyoxalase system, we performed an in vitro assay using recombinant GLO1 and found GLO1 is capable of using alkMG as a substrate, generating alkLactoylglutathione (Figure S2). The rate of GLO1 conversion of the hemithioacetal of alkMG was 3−4fold higher than observed for MG. Collectively, these results demonstrate that alkMG is cell-permeable, forms hemithioacetals with glutathione, and is metabolized to alkLactate by the glyoxalase system, and the product is transported out of the cell following detoxification. Second, the high s/n ratio of alkLactate and the simplicity of the chromatogram verifies the advantage of using alkyne tagged probes for quantitation of known or discovery of novel (probe-based) metabolites as, in principle, only peaks from metabolites originating from alkMG are present, as all other endogenous blood metabolites are

mechanism, and thereby pathophysiology, of diabetic complications.12 In a pioneering study, we recently succeeded in elucidating both the blood metabolism and plasma protein post-translational modifications of a newly discovered metabolite.13 The key to success was to use an alkyne-labeled metabolite analogue (probe) that allowed us to isolate both modified proteins as well as elucidate its blood metabolism. Previously, we have also reported the use of an alkyne-labeled MG probe (alkMG) to identify AGEs on human serum albumin (HSA). These AGEs were identified on the same arginine residues as those targeted by native MG (Figure 1). Furthermore, we have demonstrated using fluorescent labeling that alkMG dosedependently reacts with proteins in cell lysates.14 Considering the importance of the endogenous MG flow, i.e., enzymatic detoxification versus protein AGE formation, we were now interested in examining whether alkMG in general behaves as a suitable MG proxy, and our original approach could be used for simultaneous monitoring of these processes in biological systems. Furthermore, we wanted to elucidate whether the plasma and erythrocyte proteins susceptible for post-translational modifications could be isolated and the individual binding sites identified.

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RESULTS AND DISCUSSION alkMG Behaves as a Methylglyoxal Proxy. MG is a small cell permeable metabolite that is primarily metabolized by the endogenous glyoxalase systems into D-lactate. This conversion is initiated by a nonenzymatic reaction between glutathione and MG giving a hemithioacetal that subsequently is rearranged by GLO1 into lactoylglutathione and finally hydrolyzed to D-lactate and glutathione by GLO2. Indeed, it is estimated that the majority (>99%) of MG is reversibly bound to glutathione and other thiols in a biological system.15 To verify glutathione hemithioacetal formation and GLO1/2 metabolism, we spiked 5 mM MG into a freshly drawn human blood sample and, following removal of blood cells and plasma proteins, analyzed the remaining soluble metabolites by untargeted LC-QTOF-MS. From the base peak chromatogram C


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Figure 4. Formation of alkLactate with limited protein modification in whole blood compared to plasma demonstrating the impact of glyoxalase on methylglyoxal metabolism and AGE formation. Samples of whole blood or plasma were treated with alkMG (0, 0.25, 1, or 5 mM) for 2 h. Precipitated proteins were conjugated to rhodamine-azide while the metabolite supernatant was enriched on streptavidin resin. (A) In-gel fluorescence scan of rhodamine-labeled proteins from whole blood and plasma. (B) LC-MS detection of alkLactate in whole blood and plasma samples.

removed during workup (compare background in Figure 2A vs B). In our initial studies with alkMG, we found that alkMG primarily forms stable adducts with arginines and to a lesser extent with lysines in accordance with the literature.14 We however never performed direct comparison of reactivity between MG and alkMG and the two amino acids primarily involved in MG modifications lysine and arginine.17 Mixing equal amounts of either MG or alkMG with N-acetyl lysine however led to no observable amount of CEL by LC-QTOFMS even after 72 h of incubation. Conversely, both MG and alkMG gave a rather complex mixture of adducts with a mass corresponding to the different isomeric hydroimidazolones (alk)MG-H1, (alk)MG-H2, and (alk)MG-H3 (Figure S3) when mixed with N-acetyl arginine.18 Simultaneously, an adduct with a mass corresponding to the known hydrolysis product of (alk)MG-H3, carboxyethylarginine (alk)CEA, was observed in the experiments (Figure S3). The combined peak areas of all (alk)MG-H-isomers were plotted together with a plot of (alk)CEA peak areas. These data indicate that the reactivity of alkMG is comparable to MG; however, formation of alkCEA, i.e., hydrolysis of alkMG-H3, perhaps is slightly slower than hydrolysis of MG-H3. In previous experiments, we also examined the site-specific modification of human serum albumin incubated with MG or alkMG and observed a similar pattern and type of modification.14 To extend these data, we performed an analogue experiment on human hemoglobin. The results showed a high correlation between sites and modification types using the two compounds, i.e., all modification sites observed with alkMG were also observed using MG. A number of further arginine and lysine modification sites could be detected with MG (Figure S4). Curiously, these sites could also be identified with the alkMG probe if the sample was enriched using click-chemistry and streptavidin beads prior to trypsination and LC-MS analysis (Figure S5). Simultaneous Profiling of alkMG Metabolism and Protein Modifications in Human Blood and Plasma.

Knowing that alkMG metabolism and protein reactivity resemble native MG, we sought to gain a global picture of both processes using the workflow depicted in Figure 3. For these studies, a recently commercialized azido-azo-biotin linker was used as a substitute for our in-house developed cleavable solid-phase resin, so only the probe would need to be synthesized in-house.16 The incubation time of alkMG was set to 2 h based on an initial evaluation of alkLactate formation in human blood where a plateau was reached after 120 min, indicating full conversion of the 1 mM spike of alkMG (Figure S7). To evaluate the impact of alkMG glyoxalase metabolism on the degree of protein modification, an experiment in blood plasma was performed in parallel. In brief, freshly drawn blood samples from healthy subjects were divided into two aliquots: one was used directly, and one was centrifuged to remove blood cells from the plasma. Blood and plasma samples were then incubated with 0 (control), 0.25, 1, or 5 mM alkMG for 2 h at 37 °C. Following incubation, blood cells were separated from the whole blood samples, and alkMG modified plasma proteins from all samples were precipitated and conjugated to rhodamine-azide (Figure 3 middle). Proteins were then separated using SDS-PAGE, and alkMG modified species were visualized using in-gel fluorescence (Figure 3A). The alkMG treated whole blood samples did not show any detectable modification at 0.25 and 1 mM. In accordance with our earlier preliminary studies, only at 5 mM alkMG probe was a weak band observed at 65 kDa, a mass corresponding to the major plasma protein albumin. Similarly, only a weak labeling of intracellular proteins in erythrocytes has been observed.13 Conversely, the cell-devoid plasma samples showed a much more significant degree of protein modification, with bands observable at the lowest concentration tested (0.25 mM, Figure 4A). This different pattern of protein labeling, depending on the presence of blood cells, suggests that alkMG efficiently can modify plasma proteins. Further, this indicates the importance of blood cell MG metabolism on the degree of plasma protein modification. We D


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Table 1. Proteins and Their Corresponding alkMG-H1/alkCEA or alkCEL Modification Sites Detected in Plasma Treated with 250 μM alkMGa protein alpha-1-antichymotrypsin alpha-1-antitrypsinb alpha-1B-glycoprotein alpha-2-antiplasmin alpha-2-HS-glycoproteinb alpha-2-macroglobulin antithrombin-III apolipoprotein A-Ib apolipoprotein A-IV apolipoprotein B-100b apolipoprotein C-Ib apolipoprotein C-III apolipoprotein L1 CCA tRNA nucleotidyltransferase 1, mitochondrial cell division control protein 6 homolog clusterin (ApoJ)b complement C3b fibrinogen alpha chainb fibrinogen beta chainb fibrinogen gamma chain haptoglobin-related proteinb hemopexin histidine-rich glycoprotein Ig alpha-1 chain C regionb interalpha-trypsin inhibitor heavy chain H2 interalpha-trypsin inhibitor heavy chain H4b kininogen-1b laminin subunit gamma-2 methylcytosine dioxygenase TET1 nuclear body protein SP140-like protein plasminogen; plasmin heavy chain Ab protein AMBP secretoglobin family 1D member 2 prothrombinb sentrin-specific protease 3 serotransferrin serum albumin serum amyloid A-4 protein vitronectinb

gene SERPINA3 SERPINA1 A1BG SERPINF2 AHSG A2M SERPINC1 APOA1 APOA4 APOB APOC1 APOC3 APOL1 TRNT1 CDC6 CLU C3 FGA FGB FGG HPR HPX HRG IGHA1 ITIH2 ITIH4 KNG1 LAMC2 TET1 SP140L PLG AMPB SCGB1D2 F2 SENP3 TF ALB SAA4 VTN

alkCEL position(s)

alkMG-H1/alkCEA position(s) 350 220, 305, 306 254 81 337 715, 719, 732, 1031, 1401 78, 79, 177, 43123 51, 85, 107, 140, 147, 155, 173, 177, 184, 195, 201, 212, 2399 275, 279, 326 2112,242133, 3212 49, 54, 65 60 303 312

338

314 269

215, 336, 425 161, 304, 315, 740, 748, 880, 881, 954 38, 129, 137, 160, 216, 218, 258, 263, 271, 287, 425,426, 512, 573, 59125 53, 60, 72, 196, 199, 206, 42125 134 10326 83, 89,26 213, 219, 222, 378 111 273 464 56, 500, 616, 644, 649, 898 187, 308, 392 1062 938 277 523,27 663 140 74 436

52

267

505, 506 132, 327, 343, 663 34, 141, 169, 210, 233, 242, 246, 434, 452, 496, 50928 120 330, 453

a Sites of modification that have been previously reported to be adducted by MG are identified in bold. Amino acids are numbered according to their Uniprot sequence. Identified sites were determined using MaxQuant as either alkMG-H1 (Arg +277.1117) or the precursor alkCEA (Arg +288.1222) or alkCEL (Lys +288.1222). Selection criteria for the sites are given in Star Methods under Data Analysis of alkMG Binding Site Peptides from Plasma. Positions highlighted in bold have been previously identified as modified by MG in vitro or vivo. bAnnotated protein hits where the experimental data also match additional homologous protein sequences (see Table S1 for complete list).

Identification and Profiling of Plasma alkMG Targets. Identification of proteins and individual amino acid residues prone to AGE formation in biological matrices was a primary reason for developing the alkMG probe. To investigate this, we utilized the previously prepared plasma samples as the degree of protein modification was significantly greater in the absence of glyoxalase activity. Following the workflow depicted in Figure 3 (bottom), an aliquot of alkMG labeled plasma proteins was conjugated to the azido-azo-biotin linker, digested, and enriched using streptavidin beads. After chemical cleavage, modified peptides were analyzed using nanoLC-MS/ MS. This analysis yielded a significant number of labeled plasma protein binding sites in each treatment group (Table 1

next analyzed the soluble metabolite fractions for alkLactate. As observed in Figure 2, the major peak detected in the chromatograms from the whole blood samples was alkLactate. Interestingly, a dose-dependent increase of alkLactate up to the highest level of probe (5 mM) applied was observed (Figure 4B). In contrast, negligible formation of alkLactate (and no other major metabolite) was observed in the experiments with the cell-devoid plasma samples. These observations indicate the very high efficiency of the blood cells and the glyoxalase system in detoxifying glyoxals in vivo. Collectively, these findings confirm that this procedure is an efficient means to monitor both alkMG metabolism and protein modification simultaneously. E


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Figure 5. Dose- and time-dependent alkMG labeling of erythrocyte lysates. Samples of erythrocyte lysate were incubated with alkMG (0, 0.1, 0.5, or 1 mM) for 2, 8, or 24 h. Precipitated proteins were conjugated with rhodamine-azide for in-gel fluorescence scanning according to workflow Figure 3 (middle).

Table 2. Proteins and Their Corresponding alkMG-H1/alkCEA and alkCEL Modification Sites Detected in Erythrocyte Lysate Treated with 0.5 mM alkMGa protein

gene

alkMG-H1/ alkCEA positions

actin cytoplasmic 2b adenylate kinase isoenzyme 1 ankyrin-1 band 3 anion transport protein carbonic anhydrase 1 carbonic anhydrase 2 catalase

ACTG1 AK1 ANK1 SLC4A1 CA1 CA2 CAT

erythrocyte band 7 integral membrane protein flavin reductase (NADPH) fructose-bisphosphate aldolase a glyceraldehyde-3-phosphate dehydrogenase hemoglobin subunit alpha

STOM

183, 206, 254 53, 107 426 146, 150, 292, 346 77, 247, 258 27 68, 127, 130, 252, 363, 382, 431, 458 8, 215, 232

BLVRB ALDOA GAPDH

92, 134, 140, 174 56, 57 200

hemoglobin subunit beta hemoglobin subunit beta or delta hemoglobin subunit delta hemoglobin subunit gamma-1 hemoglobin subunit gamma-2 L-lactate dehydrogenase B chain NF-kappa-B inhibitor delta nucleoside diphosphate kinase Bb peptidyl-prolyl cis−trans isomeraseb peroxiredoxin-1 peroxiredoxin-2 peroxiredoxin-2b

HBA1

32, 9331−33

HBB

31, 4131

HBB or HBD HBD HBG1 HBG2 LDHB NFKBID NME2 PPIAL4C PRDX1 PRDX2 PRDX2

alkCEL positions

gene

peroxiredoxin-6 POTE ankyrin domain family member Eb protein 4.1 protein S100-A4 Hsc70-interacting proteinb putative RNA-binding protein 15B rabenosyn-5 solute carrier family 2, facilitated glucose transporter member 1 spectrin alpha chain, erythrocytic 1 spectrin beta chain, erythrocytic

PRDX6 POTEE

22, 53, 155 954

EPB41 S100A4 ST13 RBM15B RBSN SLC2A1

375 40, 49 271 188 395 458

spectrin beta chain, nonerythrocyticb triosephosphate isomerase tropomyosin alpha-3 chainb vesicle transport through interaction with t-SNAREs homologue 1A

8, 12, 17, 41 9, 60, 62, 96 67, 14534 96

145 145 100 183 88 37

alkMG-H1/ alkCEA positions

protein

SPTA1 SPTB SPTBN1 TPI1 TPM3 VTI1A

alkCEL positions

28, 362, 785, 855, 1262, 1493, 1735 477, 840, 1010, 1423, 1737, 1746 425, 406, 2050 137 92 127, 130

a Positions in bold indicate sites that have previously been identified as targets of MG-H1 modification by MG in vivo or vitro. Amino acids are numbered according to their Uniprot sequence. Identified sites were determined using MaxQuant as either alkMG-H1 (Arg +277.1117) or the precursor alkCEA (Arg +288.1222) or alkCEL (Lys +288.1222). Selection criteria for the sites are given in Star Methods under NanoLC-MS/MS Detection and Data Analysis. Positions highlighted in bold have been previously identified as modified by MG in vitro or in vivo. bAnnotated protein hits where the experimental data also match additional homologous protein sequences (see Table S2 for complete list).

151 109, 110, 127, 139, 150 66

lesser extent, the CEL modification on lysine residues.20 In recent studies, a precursor to MG-H1 formally represented as a MG-H3 hydrolysis product carboxyethylarginine (CEA)

and Table S1). In vivo, MG takes part in a number of different post-translational modifications, with the quantitatively most abundant being the MG-H1 modification of arginine and, to a F


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ACS Chemical Biology has also been found in high abundance.18,21,22 A similar pattern was observed using our probe, where we primarily detected a mass increase corresponding to alkMG-H1 (Figure S8) or the precursor alkCEA, whereas the mass increase corresponding to alkCEL (Figure S9) was less abundant and primarily detected at higher concentrations of the probe (for structures and corresponding mass addition see Figure S10). In total, we detected 229 different alkMG-modified peptides from 62 different proteins. A total of 127 alkMG modified peptides, corresponding to 39 different proteins, were detected at the lowest concentration of the MG probe tested (250 μM). alkCEA was furthermore detected at most of these alkMG-H1 sites in an average alkMG-H1: an alkCEA intensity ratio of 2:1 based on the crude peptide signal. This is in line with recent data pointing at CEA as a major MG derived AGE at levels comparable to MG-H1.22 Additionally, we detected seven alkCEL modification sites at 250 μM and one more at higher concentrations. The modified peptides detected at 250 μM are reported in Table 1. The full list of modifications found at all concentrations of probe can be found in Table S1. Identification and Profiling of alkMG Targets in Erythrocyte Lysate. For a more complete profiling of alkMG targets in human blood, we also investigated erythrocyte protein modifications. Erythrocytes constitute the most abundant cell type in blood, with a main function as an oxygen carrier. Erythrocytes are primarily glycolytic and have high basal levels of MG, which is exacerbated in diabetes patients.29 Erythrocyte-derived glycated hemoglobin (HbA1c) is currently the gold standard for diagnosing diabetes.30 On the basis of the high glyoxalase activity in intact erythrocytes against our alkMG probe, lysates were used for profiling experiments. In brief, erythrocytes were isolated from freshly drawn human blood samples by centrifugation and lysed prior to alkMG treatment (0 (control), 0.1, 0.5, or 1 mM alkMG for 2, 8, or 24 h). After incubation, proteins were precipitated and reacted with rhodamine-azide to investigate the levels of labeling using in-gel florescence. As shown in Figure 5, a doseand time-dependent response was observed. On the basis of these data, a new set of erythrocyte lysate samples was treated with alkMG to a final concentration of 0 (control), 0.5, or 1 mM, incubated for 24 h at 37 °C to be analyzed for site-specific modifications, as described. These analyses revealed a significant number of modification sites identified at both levels of alkMG studied. In total, 120 alkMGmodified peptides were detected from 46 different proteins (Table S2). As expected, modified peptides of hemoglobin were identified; in addition, numerous proteins with structural, metabolic, antioxidative, or transport functionalities were also identified. From these, 91 modified peptides of 36 protein hits were detected at the lowest concentration of the alkMG probe tested (0.5 mM) (Table 2). As for the plasma proteins, the quantitatively most abundant modification type was MG-H1 on arginine residues, and alkCEA was again detected at most of the alkMG-H1 sites. To evaluate our protocol further, a comparison was made between alkMG (and MG) modification sites detected on erythrocytic hemoglobin with sites detected using MG and alkMG on isolated human hemoglobin. In brief, hemoglobin was treated with native MG or alkMG, at concentrations of 0 (control), 0.5, or 1 mM for 24 h at 37 °C. Following tryptic digestion (MG) or click-chemistry-based workup (alkMG) and trypsination, the resulting peptides were analyzed by nanoLCMS/MS. Many modifications were found to be identical

between the three quite dissimilar setups, and of particular relevance all MG AGE sites reported in the literature were identified by the alkMG probe. Application of the alkMG protocol on isolated hemoglobin furthermore gave a number of additional sites not found in the lysate, indicating that further data can be obtained in the future following method improvements (Figure S5). Simultaneous Studies of alkMG Protein Modifications and Metabolism in Cell Culture. For a further test and validation of our method, we finally included an investigation of metabolism and intracellular protein modifications in a functionally more complete HeLa cell line. These cells have previously been used to study the impact of GLO1 inhibitors on MG metabolism and cancer cell survival.35 Following the workflow in Figure S11, HeLa cells were treated with 0 (control), 1, or 5 mM alkMG for 2 h. The cells were lysed and proteins precipitated and analyzed using SDS-PAGE and fluorescent scanning, whereas cell culture media and the supernatant from the protein precipitation step, both potentially containing metabolites, were analyzed by LCQTOF-MS. The fluorescence scan revealed a high degree of intracellular protein modification using 5 mM alkMG probe, whereas a fainter signal was observed at 1 mM alkMG (Figure S12A). Apparently, the glyoxalase system is not able to keep up with the concentration of alkMG at 5 mM, leading to a higher degree of protein modification. This mechanism is supported by the LC−MS analysis of the enriched metabolites, which shows alkLactate as the primary metabolite at both 1 and 5 mM in both the cell lysate and the culture medium (Figure S12B). alkMG as a Methylglyoxal Proxy. We have previously demonstrated that alkMG reacts with human serum albumin (HSA) forming alkMG derived advanced glycation products with similar reactivity and selectivity as native MG. These data we expanded to also include hemoglobin and found that all sites detected using alkMG are confirmed using MG, though several further sites were discovered using MG. From experiments using N-acetyl arginine, it furthermore appears as the reactivity of alkMG is at least as high as MG, pointing to the difference between modification sites between the two compounds maybe being due to steric differences, e.g., the introduction of the alkyne group in alkMG. The sites that escaped detection using alkMG and analysis following direct trypsination of hemoglobin however were found following click-chemistry and enrichment prior to trypsination and LCMS analysis, demonstrating that alkMG in combination with the enrichment procedure can elucidate the same modifications as found using MG in vitro. The remaining aspects to validate that alkMG is a reasonable proxy for MG were to demonstrate that alkMG also replicate the general metabolic behavior of MG in human whole blood. In particular, we discovered that alkMG undergoes a blood-cell-dependent metabolism, demonstrating that alkMG most likely diffuses into the blood cells where it almost exclusively undergoes GLO1 and -2 catalyzed rearrangement to alkLactate. Following this transformation, a part of alkLactate is excreted back into the blood plasma. That alkMG is a substrate for GLO1 was further verified in vitro with a dedicated GLO1 activity assay. The reactivity of alkMG toward GLO1 catalyzed isomerization was higher than for MG, which is in line with observations made using the related 4,5-dioxovalerate as a substrate.36 Though we did not quantify the exact amount of alkLactate formed, we used a global UPLC-QTOF-MS based profiling G


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post-translational modifications play an important role in the deleterious effect of MG on cell health; the detailed mechanisms through which these are exerted, however, remain unknown.4,42,43 In relation to plasma proteins, MG has been shown to affect both HDL levels and blood coagulation.4,23,25,44 In our investigation into alkMG binding sites in plasma, we have identified a broad range of sites on plasma proteins that may mimic those found in vivo. In accordance with the literature, the major type of modification detected is the arginine derived alkMG-H1 as well the tentative precursor alkCEA. Although preliminary research has been carried out primarily using exogenous MG in vitro (vide inf ra), such an indepth study of potential MG targets in plasma has, to the best of our knowledge, not been performed. The abundant plasma proteins, such as fibrinogen, apolipoprotein A1, and serum albumin, were found to have the most modification sites in our study. In a previous study on albumin, five arginine residues (Arg138, Arg210, Arg242, Arg434, and Arg452) were found to be modified in vitro at a concentration of MG of 500 μM.28 We detect 11 modified arginine residues at 250 μM alkMG, with four of them corresponding to those previously reported. Another recent study detected nine modified arginine residues in albumin from plasma treated with 500 μM MG, with five also found in our study in the 250 μM samples. 26 Apolipoprotein A1 was found to be highly modified at the lowest concentration of the MG probe, reacting at 13 sites at 250 μM among those Arg51, Arg147, and Arg173. The same sites are known to be modified in vivo by MG and increasingly in diabetic patients compared to controls. The modifications furthermore suggested to have a functional effect on highdensity lipoprotein (HDL) degradation and functionality in vivo.44 Apolipoprotein B-100 was found to be modified at three sites. This protein is also known to be modified by MG in vivo, and its modification might be involved in cardiovascular diseases.24,45 The most heavily modified protein in our studies is fibrinogen, with 15 modifications on the alpha chain, seven on the beta chain and one on the gamma chain. This tendency is in accordance with a recent in vitro study using MG.25 We also detected two modifications of plasminogen at 250 μM probe, where one (at Arg523) was reported previously under in vitro conditions.27 Modification at this residue might decrease ligand affinity in the binding site. Antithrombin-III was detected with four binding sites in our study. This protein was reported to be an in vitro target of MG in a previous study, where modification was shown to impair its function as an anticoagulant.23 The authors of that study only provided evidence for the active-site residue Arg425, on which they detected the modification. The modification was not detected on the active site in our analysis; however, one modification (at Arg177) that we detected was at a residue that is part of the heparin-binding site, and modification here could likely also inhibit antithrombin-III activity. Thus, there could be a dual mode of inhibition by MG on antithrombin. We further detected modifications on serotransferrin, haptoglobin, and hemopexin, which have already been described as targets of MG in treated plasma but at different sites than the ones we identified.26 A number of other proteins that we reliably detected at the 250 μM MG probe level have never been identified as MG targets, such as alpha-1-antitrypsin, vitronectin, prothrombin, and clusterin (Apolipoprotein J). Alpha-2-macroglobulin was found to be modified at five sites, with three (Arg715, Arg719 and Arg732) being in the inhibitory regions of the protein, potentially affecting its

method to evaluate formation of alkMG derived products, and from the chromatogram it was directly evident that only one major product, alkLactate, was formed in the blood samples. Similar studies were conducted in cell culture, where parts of alkLactate were also detected in the cytoplasm, all validating that alkMG is a suitable MG proxy. A Novel Tool for Simultaneous Monitoring of Methylglyoxal Metabolism/Glyoxalase Activity versus Protein AGE-formation. Our work has identified a method and workflow for simultaneous profiling of alkMG detoxification vs formation of alkMG-derived AGEs. Endogenous plasma concentrations of MG are in the range of 50−150 nM for healthy individuals and correspondingly higher for those with unregulated diabetes.33 Our first profiling data showed that even by spiking levels of alkMG up to 5 mM in a whole blood sample (100 000× endogenous plasma levels of MG), only limited plasma or intracellular protein modifications were evident. This most likely reflects a very high efficiency of the endogenous glyoxalase systems in detoxifying alkMG and MG. These findings were supported by the metabolite profiling data that showed an almost linear dose−response relationship between levels of alkMG and alkLactate, validating the glyoxalase system as the dominant route of alkMG-flux. In the absence of any blood cells and glyoxalase activity, negligible formation of alkLactate and robust plasma protein modification was observed. Similar results were obtained in cell culture, where alkLactate was profiled both in culture media as well as in cell lysate. On the basis of these results, it should be possible to develop robust methods to screen for chemical modifiers of glyoxalase activity via the measurement of alkLactate. Traditional LC-MS based methods require the separation of D- and L-lactate, which proves challenging; our method measuring alkLactate as a Dlactate proxy provides a simpler approach.37 In this connection, more compounds have recently emerged as possible GLO1 inducers, some with clinical relevance.38 Along the same line, dysfunctional MG metabolism has been linked to the development of insulin resistance and diabetes. Indeed, 15− 25-fold higher intracellular levels of MG have been detected in erythrocytes from diabetics compared to controls.29 Evidence points to an increased formation of glycolytic intermediates the direct precursors to MGas the cause. By applying alkMG to blood samples or isolated erythrocytes from diabetics, it should be possible to investigate glyoxalase activity and simultaneously profile if alternative routes of MG detoxification are operative in erythrocytes from diabetic patients (vide infra). Simultaneously, we can profile how protein AGE formation is influenced in the diabetic erythrocytes or whole blood samples. Such investigations would also be relevant to help illuminate why GLO1 knockout in a Drosophila model only slightly (50%) changes endogenous levels of MG, whereas in C. elegans knockout of the GLO1 homologue glod4 apparently increases levels of MG more than 1000-fold.39,40 Of relevance, CRISPR/Cas9 based knockout of GLO1 in mammalian Schwann cells was shown to induce compensatory mechanisms to detoxify MG, metabolism that possibly also could be traced with the alkMG probe combined with the untargeted LC-QTOF analysis as applied in the method.41 Plasma Protein Targets for alkMG Reflect Known AGEs and Directs Further Research. Of equal importance to profiling of metabolism and AGE formation, is insight into which proteins are susceptible to alkMG modifications as well as their individual modification types and sites. MG-mediated H


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NAD+.52 We find this amino acid to be modified, which likely affects the enzymatic activity. In addition, inhibition of LDH by MG has been shown in vitro.51 Another enzyme, nucleoside diphosphate kinase B, also contains a central arginine residue (Arg88) important for the coordination of phosphates in ATP.53 This arginine residue is found to be modified by alkMG in our study. Furthermore, flavin reductase (NADPH) contains a central arginine residue (Arg132) that coordinates to NAD+.54 We identified Arg134 to be modified, and due to the proximity to Arg132, this modification might affect the coordination to NAD+. However, functional studies on the impact of MG have not yet been performed. Carbonic anhydrase catalyzes the conversion of carbon dioxide to bicarbonate and hydrogen ions, which affect the pH of the blood. The activity of carbonic anhydrase has been reported to increase in a dose-dependent manner when incubated with MG.55 However, whether our identified modifications of Arg77, Arg247, Arg258, and Arg27 have functional effects is not known and needs further research. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen. Thereby, it protects cells from cellular oxidative stress. In our study, catalase is found to be modified at eight sites (Arg68, Arg127, Arg130, Arg252, Arg363, Arg382, Arg431, Arg458). From these, only Arg68 has previously been reported as modified by MG in studies of bovine catalase; however, the activity was not found to be affected.56 Similarly to the plasma proteome, it generally seems that erythrocyte proteins reported to be targets for MG modifications or implicated in MG related cellular dysfunction in the literature are also targeted by alkMG in our studies. The relatively few known MG reactive sites could mostly be verified by our probe, and in addition our study points to hitherto unknown alkMG/MG reactive arginines that potentially could be central to the observed functional effects. Furthermore, in our studies, we identify a relatively large number of unknown and potential protein targets to advanced glycation, pointing to new directions for elucidating the role of MG in biology. General Use of Alkynylated Probes for Simultaneous Monitoring of Metabolism and Protein Modification. An increasing amount of evidence accumulates on small metabolites having both roles as energy metabolites and simultaneously as entities that either directly or via posttranslational modifications regulate protein or enzyme activity or function.1−4 For some of these metabolites, alkynylated probes already exist, and for a selection of these it is even known that they are accepted as substrates by the endogenous enzymesa prerequisite to being useful as a probeand undergo a range of catalytic transformations before they are anchored at different proteins or enzymes.57−61 By using our procedure, it is possible to get further detailed insight into their metabolism, if it is not completely elucidated, or measurements of metabolite concentrations/flux concomitantly with protein post-translational modifications as we have done with alkMGalkLactate. A relatively simple tool for simultaneous elucidation of novel metabolites and protein adduction will also be of particularly high importance in the drug-discovery process, as stable metabolites might elicit unwanted biological effects and toxic protein reactive metabolites usually are difficult to monitor.62−65 Further studies along these lines are in progress.

function as a protease inhibitor. Complement C3 was also detected at the lowest concentration of probe used, with modifications at eight sites. The Arg748 residue is of particular interest, as it is the site cleaved by C3 convertase and is essential for the function of the complement system in the immune response.46 Erythrocyte Protein Targets for alkMG Reflect Known AGEs and Directs Further Research. In our study of alkMG-modification sites in erythrocytic proteins, we have identified a broad range of sites on proteins with the major modification type being alkMG-H1 and alkCEA similarly to the plasma proteins. The more abundant erythrocytic proteins, hemoglobin, carbonic anhydrase, peroxiredoxin, spectrin, and catalase, were found to have the most modification sites. In previous studies of hemoglobin, MG has been reported to modify Arg32, Arg93, and Arg142 in the α chain and Arg31, Arg41, and Arg105 as well as Lys67 and Lys145 in the β chain. We also identified these modification sites, but also additional CEL modifications that, to the best of our knowledge, have not been reported. However, in contrast to the previous reports, we did not find alkMG to modify Arg142α nor Arg105β even though the latter is the most accessible to solvent, hence less hindered for reaction.32,47 This may be explained by influence from amino acids in the microenvironment surrounding the individual amino acid residues affecting the reactivity toward (alk-)MG. Although Arg105β is most accessible, it might not be the most reactive to MG, under the applied conditions in this study. When modified, the structural and functional properties of hemoglobin have been reported to be affected, disturbing its reactivity. These structural and functional alterations include an increase in α-helical content, altered thermostability, increased iron release, increased peroxidase activity, and decreased esterase activity.32 Several metabolic enzymes were found with numerous modifications in our study, e.g., fructose-bisphosphate aldolase A (Arg56 and Arg57), triosephosphate isomerase (Arg137), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Arg200). These enzymes are central in glycolysis, and thus involved in the formation of MG. In this pathway, fructose-bisphosphate aldolase A is catalyzing the formation of the two direct MG precursors (triosephosphate intermediates), GA3P and DHAP, from fructose-1,6-biphosphate. In vitro studies of fructosebisphosphate aldolase A have indeed shown MG to decrease its enzymatic activity.48 This might be explained by the discovered modification of Arg56, a conserved residue which is important for binding the anionic C-1 phosphate of the substrate.49 Further, highly increased levels of GA3P and DHAP have been detected in erythrocytes isolated from in diabetic patients.50 In the studies of diabetic patients, a reduced activity of GAPDH was also observed while in vitro studies of GAPDH have shown a time- and dose-dependent inhibition when incubating with MG, both for the isolated enzyme and in cell lysates.51 As a result, the flux through the glycolysis might become reduced under these conditions. However, no specific modification sites by MG have, to the best of our knowledge, been reported for these enzymes up to now. Additionally, a subgroup of enzymes catalytically dependent on central arginines in the anionic coordination of phosphates in either NAD+ or ATP was identified in our study. An example is the metabolic enzyme Llactate dehydrogenase (LDH) that, under anaerobic conditions, catalyzes the stereospecific reduction of pyruvate to lactate in order to regenerate NAD+. In this enzyme, Arg100 is a part of the active site loop important for binding the cofactor

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METHODS

Measurement of GLO1 Activity. GLO1 activity was quantified via incubation of purified GLO1 (1.5 ng; R&D Systems, Minneapolis, I


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μL of a 3:7 mixture of 50 mM CuSO4 and 100 mM THPTA, and 50 μL of 100 mM α-aminoguanidine. After vortexing, the click reaction was initiated by the addition of 50 μL of freshly prepared 100 mM sodium ascorbate and vortexed again. Samples were reacted for 1 h at rt, vortexed after 30 min, and then added to 100 μL of 0.5 M EDTA to quench the reaction. The proteins were precipitated by MeOH/ CHCl3, centrifuged (7000g, 5 min, 4 °C), and washed in 500 μL of MeOH/H2O (9:1) before another centrifugation. The pellets were air-dried and redissolved in 400 μL of 6 M urea. All samples were diluted to 500 μL with a digestion buffer. The samples were reduced by adding a freshly made 1 M DTT solution to a final concentration of 10 mM and incubated 30 min at rt, then alkylated by adding freshly prepared iodoacetamide to a final concentration of 25 mM and incubating 1 h at rt in dark. The alkylation reaction was quenched with 15 μL of 0.5 M DTT. All samples were diluted with 800 μL of digestion buffer (∼2 M urea final concentration). Trypsin (1:40) and 2 μL CaCl2 (200 mM) were added, followed by incubation at 37 °C for 16 h with shaking. A total of 100 μL of a 50% suspension of streptavidin agarose resin was added, and the samples were incubated for 1 h on a rotating mixer. The resin was collected by centrifugation (2000g, 2 min, rt), and the supernatants were carefully removed. The resin was washed with 2× 1% SDS in PBS (1 mL), 2× 0.5% SDS in PBS (1 mL), and 2× digestion buffer (1 mL). The binding site peptides were then eluted twice by incubation with 75 μL of elution buffer (50 mM Na2S2O4/digestion buffer) for 30 min each at rt with shaking. The resin was washed with 50 μL of digestion buffer, which was combined with the elutions. The samples were acidified with 15 μL of 10% formic acid to pH ∼ 2 and purified using mixed-cation exchange chromatography columns (Waters) and concentrated using vacuum centrifugation before nanoLC-MS/MS analysis.

MN) with a master mix of 2.5 mM GSH and 2.5 mM MGO (preincubated at 37 °C for 1 h to generate hemithioacetal substrate) at 37 °C for 30 min. A total of 1 nmol of internal standard (GSH(glycine-13C2,15N)) was then added to each sample, and reactions were immediately quenched via the addition of 200 mM ethyliodoacetate (20 mM final concentration). Samples were derivatized for 30 min at RT protected from light with end-over-end mixing. Protein was precipitated via the addition of 20% (w/v) 5-sulfosalicylic acid (2% final) and removed via centrifugation at 10 000g for 5 min at RT. Supernatants were removed and diluted 1:20 in H2O containing 200 mM heptafluorobutyric acid (HFBA).66 Clarified supernatant (5 μL) was chromatographed using an Agilent LC system equipped with a 50 × 2.1 mm, 2.6 μm particle diameter Kintetix C8 column (Phenomenex, Torrance, CA) at a flow rate of 200 μL/min. Solvent A was 10 mM HFBA in H2O, and solvent B was 10 mM HFBA in ACN. The gradient was as follows: 0 min (1% B), 3 min (80% B), 4 min (99% B), 4.2 min (1% B), 7 min (1% B). The needle was washed prior to each injection with a buffer consisting of 25 mM NH4OAc in MeOH. Multiple reaction monitoring was performed in positive ion mode using an AB SCIEX 6500 QTrap with the following transitions: m/z 394.2 → 265.2 for GSH; 380.1 → 233.1 for LGSH; 418.1 → 271.0 for alkLGSH; 397.2 → 268.2 for GSH-(glycine-13C2,15N). GSH, LGSH, and alkLGSH were quantified using GSH-(glycine-13C2,15N) as the internal standard. Enrichment and UPLC-QTOF-MS Analysis of alkMG Metabolites (Figure 3 top). Samples of supernatants from the acetone precipitation were added to 12 μL of 5 mM azido-azo-biotin, 50 μL of a 3:7 mixture of 50 mM CuSO4 and 100 mM THPTA, and 25 μL of 100 mM α-aminoguanidine. After vortexing, the samples were added to 25 μL of freshly prepared 100 mM sodium ascorbate to initiate the click reaction. The reaction was incubated for 1 h at rt with vortexing after 30 min and then quenched with 50 μL of 0.5 M EDTA. A total of 50 μL of a 50% suspension of streptavidin agarose resin was added and left 1 h on a rotating mixer. The resin, now containing the biotinlabeled intracellular metabolites of alkMG, was collected by centrifugation (2000g, 2 min, rt). The supernatant was removed, and the resin was washed with 2× 1% SDS in PBS (1 mL), 2 × 0.5% SDS in PBS (1 mL), and 2× digestion buffer (1 mL). Between each wash, the resin was recollected by centrifugation (2000g, 2 min, rt). The resin-bound metabolites were then released by the addition of 50 μL of elution buffer (50 mM Na2S2O4 in digestion buffer) followed by incubation for 30 min at rt. This was repeated twice, and the supernatants were collected. The resin was washed with 50 μL of digestion buffer, and all the eluates were combined and frozen immediately until LC-MS analysis. Fluorescence Labeling and In-Gel Visualization of alkMG Target Proteins (Figure 3 middle). Protein samples were added to a premixed aliquot consisting of 3 μL of 10 mM rhodamine-azide, 10 μL of a 3:7 mixture of 50 mM CuSO4 and 100 mM THPTA, and 5 μL of 100 mM α-aminoguanidine and then vortexed. A total of 5 μL of 100 mM freshly made sodium ascorbate was added to initiate the click reaction before incubation for 1 h at rt, vortexing after 30 min. A total of 10 μL of 0.5 M EDTA was then added to quench the reaction. Excess reagents were removed by MeOH/CHCl3 protein precipitation and centrifugation (7000g, 5 min, 4 °C). Pellets were washed in 500 μL of MeOH/H2O (9:1) before another centrifugation. All tubes were inverted onto absorbing paper and air-dried for 5 min to avoid excessive drying of the protein pellet. Then, 50 μL of SDSPAGE loading buffer was added to each sample. The samples were treated with sonication, heated to 95 °C for 5 min, and centrifuged (14 000g, 2 min, rt). Five μL (20 μg) of each sample was loaded on a Criterion “Any kD” TGX gel. The gel was run at 220 V in a Trisglycine running buffer (0.025 M Tris/0.192 M glycine/0.1% SDS, pH 8.5), then scanned for in-gel fluorescence using an ImageQuant LAS 4000 (GE Healthcare) in green light mode. The gel was stained using standard Coomassie staining and destaining to evaluate total protein loading. Enrichment and NanoLC-MS/MS Analysis of alkMG Binding Site Peptides (Figure 3 bottom). The samples were added to a premixed aliquot consisting of 10 μL of 5 mM azido-azo-biotin, 100

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00732. Complete list of peptide sequences (XLSX) Peptide sequences (XLSX) Supporting methods and figures, experimental models, method details, quantification and statistical analysis, and chemical synthesis (PDF) Accession Codes

The proteome data sets are available via ProteomeXchange with identifier PXD011314.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

James J. Galligan: 0000-0002-5612-0680 Karl Anker Jørgensen: 0000-0002-3482-6236 Mogens Johannsen: 0000-0002-2548-7025 Author Contributions ⊥

These authors contributed equally

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.J. gratefully acknowledges support from the Velux Foundations (VELUX34148), and the Lundbeck Foundation (R180-2014-2740). J


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