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Research article Received: 8 August 2014

Revised: 30 December 2014

Accepted: 7 January 2015

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3569

Specific tandem mass spectrometric detection of AGE-modified arginine residues in peptides Rico Schmidt,† David Böhme,† David Singer and Andrej Frolov* Glycation is a non-enzymatic reaction of protein amino and guanidino groups with reducing sugars or dicarbonyl products of their oxidative degradation. Modification of arginine residues by dicarbonyls such as glyoxal and methylglyoxal results in formation of advanced glycation end-products (AGEs). In mammals, these modifications impact in diabetes mellitus, uremia, atherosclerosis and ageing. However, due to the low abundance of individual AGE-peptides in enzymatic digests, these species cannot be efficiently detected by LC-ESI-MS-based data-dependent acquisition (DDA) experiments. Here we report an analytical workflow that overcomes this limitation. We describe fragmentation patterns of synthetic AGEpeptides and assignment of modification-specific signals required for unambiguous structure retrieval. Most intense signals were those corresponding to unique fragment ions with m/z 152.1 and 166.1, observed in the tandem mass spectra of peptides, containing glyoxal- and methylglyoxal-derived hydroimidazolone AGEs, respectively. To detect such peptides, specific and sensitive precursor ion scanning methods were established for these signals. Further, these precursor ion scans were incorporated in conventional bottom-up proteomic approach based on data-dependent acquisition (DDA) LC-MS/MS experiments. The method was successfully applied for the analysis of human serum albumin (HSA) and human plasma protein tryptic digest with subsequent structure confirmation by targeted LC-MS/MS (DDA). Altogether 44 hydroimidazolone- and dihydroxyimidazolidine-derived peptides representing 42 AGE-modified proteins were identified in plasma digests obtained from type 2 diabetes mellitus (T2DM) patients. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at publisher’s web site. Keywords: AGEs; glycation; glyoxal-derived hydroimidazolone (Glarg); methylglyoxal-derived hydroimidazolones (MG-H); precursor ion scan

Introduction

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* Correspondence to: Andrej Frolov, Institut für Bioanalytische Chemie, Biotechnologisch-Biomedizinisches Zentrum, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany. E-mail: [email protected] †

These authors contributed equally in the manuscript. Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig, Leipzig, Germany

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Protein glycation is a common non-enzymatic post-translational modification.[1] This process involves reaction between reducing sugars or dicarbonyl products of their oxidative degradation (e.g. glyoxal and methylglyoxal) and lysyl or arginyl side chains of proteins.[2,3] The interaction of sugars with lysyl amino groups yields aldose- or ketose-derived early glycation productsAmadori[4] and Heyns[5] compounds, respectively. Glyoxal and methylglyoxal, in contrast, readily react with both lysyl and arginyl residues yielding advanced glycation end-products (AGEs).[6] Besides the major lysine-related products (Nε-carboxymethyland Nε-carboxyethyllysine), several glyoxal- and methylglyoxalderived arginine-related AGEs, such as glyoxal-derived hydroimidazolone (Glarg),[7] carboxymethylarginine (CMA),[8] methylglyoxal-derived hydroimidazolones (MG-H1, MG-H2 and MG-H3)[6,9] and carboxyethylarginine (CEA)[6] (Fig. 1) are well characterized. Human tissues are continuously exposed to glucose and dicarbonyls constantly yielding AGEs which can accumulate over time.[10,11] Due to the relatively low rates of AGE formation, longliving proteins (e.g. collagens and crystallins) are more affected, than the short-living ones.[10,11] Accumulation of AGEs in these proteins is drastically enhanced under conditions of persisting hyperglycemia, characteristic for diabetes altered mechanical and functional properties[13,14] and (ii) their interaction with receptors for AGEs (RAGEs) triggering the NF-κB-mediated expression of proinflammatory mellitus.[12] The pathogenic effects of AGEs are related to (i) cross-linked extracellular matrix proteins with

molecules and inflammation response.[15] These mechanisms were proposed to contribute to the pathogenesis of diabetic complications, Alzheimer’s disease and tissue ageing.[16] Most studies dealing with the analysis of arginine-derived AGEs rely on immunochemical methods, such as ELISA[17] and Western blotting,[18] though most antibodies are not specific for a single AGE. Some AGEs, such as pentosidine and argpyrimidine, can be detected and quantified by spectrofluorometry[19] with rather high sensitivity, especially when coupled to liquid chromatography, (LC LODs of several picomols).[6] However, this approach is suitable only for fluorescent AGEs. In contrast, LC-tandem mass spectrometry (MS/MS) can be applied for specific quantification of all individual AGEs after complete enzymatic hydrolysis,[20] but this strategy does not allow the assignment of correspondingly modified proteins. Such information can be revealed by fingerprinting using MALDITOF-MS.[21] Moreover, the proteomic approach was applied for the analysis of individual proteins treated in vitro with high concentrations of dicarbonyls.[22] However, for real samples, such analyses would lack sensitivity due to the low abundance of individual

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R. Schmidt et al. Peptide synthesis

Figure 1. The structures of arginine-derived AGEs: A, 1-(4-amino-4-carboxybutyl) 2-imino-5-oxo-imidazolidine (Glarg); B, carboxymethylarginine (CMA), C-E, methylglyoxal-derived hydroimidazolones Nδ-(5-hydro-5-methyl-4-imidazolon2-yl)-ornitine (MG-H1), 2-amino-5-(2-amino-5-hydro-5-methyl-4-imidazolon -1-yl)pentanoic acid (MG-H2) and 2-amino-5-(2-amino-4-hydro-4-methyl-5imidazolon-1-yl)pentanoic acid (MG-H3), respectively; F, carboxyethylarginine (CEA).

glycated peptides and pronounced matrix-effects. For this reason, this strategy was not so far effectively applied for in vivo studies. Hence, specific methods for fast and sensitive screening of specific AGE-modified peptides are still missing. Here we present a strategy for rapid detection and identification of arginine-derived AGEs in complex enzymatic protein digests based on the detailed characterization of fragment ions formed by collision-induced dissociation (CID). Specifically, a sensitive precursor ion scan using AGE-specific reporter ions was established for Glarg- and MG-H peptides and successfully applied to glycated human serum albumin (HSA).

Experimental procedures

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Chemicals and reagents were obtained from the following manufacturers: Iris Biotech GmbH (Marktredwitz, Germany): 9-Fluorenylmethoxycarbonyl (Fmoc-) Rink Amide AM resin and Nα-Fmoc-Nω-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)L-arginine (Fmoc-L-Arg (Pbf)-OH, peptide synthesis grade), Nαallyloxycarbonyl (Alloc)-protected ornithine (Fmoc-Orn(Alloc)-OH); ORPEGEN Pharma (Heidelberg, Germany): all other Fmoc-protected L-amino acids (peptide synthesis grade); Carl Roth GmbH & Co KG (Karlsruhe, Germany): trifluoroacetic acid (TFA, 99.9%), sodium dodecyl sulphate (SDS, ≥ 99.5%); glycerol (≥99.5%), dithiothreitol (≥99%) and D-glucose hydrate (≥99.5%); Biosolve (Valkenswaard, NL): acetonitrlile (MS grade), N,N’-dimethylformamide (DMF, ≥99.8%), piperidine (≥99.5%), and dichloromethane (DCM, ≥99.9%); AppliChem GmbH (Darmstadt, Germany): Tris-HCl (ultrapure); SERVA Electrophoresis (Heidelberg, Germany): glycine (p.a.) and sucrose (>98%); Promega GmbH (Mannheim, Germany): trypsin (premium grade); VWR International GmbH (Dresden, Germany): diethyl ether (100%) and acetonitrile (≥99.9%). All other chemicals and RP-SPE, Discovery DSC-18, 1-mL cartridges were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Water was purified in house (resistance 18 mΩ/cm) on a PureLab Ultra Analytic System (ELGA Lab Water, Celle, Germany).

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Peptides 1–6 (Table 1) and Glu-1-fibrinopeptide B (H-EGVNDNEEGFFSAR-OH) were synthesized on a multiple peptide synthesizer Syro2000 (MultiSynTech GmbH, Witten, Germany) by Fmoc/tert-butyl-chemistry using eight equivalents (eq.) of Fmoc-amino acid derivatives activated with DIC/HOBt in DMF.[23] The arginine to be modified was introduced as Fmoc-Orn(Alloc)-OH. After completion of the synthesis the Alloc group was cleaved with tetrakis(triphenylphosphine)palladium(0) in the presence of N-methylmorpholine.[24] The deprotected Orn-residue was modified by reported protocols with slight modifications (Supporting information, Protocols S1 – S6).[6,25,26] Peptides were cleaved in two subsequent steps with TFA (1 mL and 0.5 mL, respectively) containing 12.5% (v/v) of a scavenger mixture (ethandithiole, m-cresole, thioanisole and water, 1:2:2:2) at room temperature (RT) in 90 min and precipitated with diethyl ether. The crude peptides were purified by RP-HPLC using water (eluent A) and 60% (v/v) aqueous acetonitrile (eluent B) containing formic acid or TFA (0.1% v/v) and a gradient slope of 1% eluent B/min.

In vitro glycation of HSA HSA (5 mg) was dissolved in 350 μL sodium phosphate buffer (pH 7.4, 0.1 mol/L) containing α-D-glucose (0.5 mol/L) and incubated at 50°C under continuous shaking (400 rpm). After seven days, the protein solution was split into 50 μL aliquots (1 mg each) and stored at 20 °C.

Table 1. Sequences of the synthetic peptides used in the tandem mass spectrometric experiments Peptide 1 1Glarg 1CMA 1MG-H1 1MG-H2 1MG-H3 1CEA 2 2Glarg 2CMA 2MG-H1 2MG-H2 2MG-H3 2CEA 3Glarg 3MG-H1 3MG-H2 4Glarg 4MG-H1 4MG-H2 5Glarg 5MG-H1 5MG-H2 6Glarg 6MG-H1 6MG-H2

Copyright © 2015 John Wiley & Sons, Ltd.

Sequence H-AGSARASGFA-NH2 H-AGSARGlargASGFA-NH2 H-AGSARCMAASGFA-NH2 H-AGSARMG-H1ASGFA-NH2 H-AGSAR MG-H2ASGFA-NH2 H-AGSAR MG-H3ASGFA-NH2 H-AGSAR CEAASGFA-NH2 H-AFGSARASGA- NH2 H-AFGSARGlargASGA- NH2 H-AFGSARCMAASGA- NH2 H-AFGSAR MG-H1ASGA- NH2 H-AFGSAR MG-H2ASGA- NH2 H-AFGSAR MG-H3ASGA- NH2 H-AFGSARCEAASGA- NH2 H-ATLRGlargETYG-OH H-ATLR MG-H1ETYG-OH H-ATLR MG-H2ETYG-OH H-LSQRGlargFPKAE-OH H-LSQR MG-H1FPKAE-OH H-LSQR MG-H2FPKAE-OH H-VSDRGlargVT-OH H-VSDR MG-H1VT-OH H-VSDR MG-H2VT-OH H-RGlargYKAAFTE-OH H-R MG-H1YKAAFTE-OH H-R MG-H2YKAAFTE-OH

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Tandem mass spectrometry for AGE detection Tryptic digest The aliquots of the glycated HSA solution or human plasma were ultra-filtrated against 0.1 mol/L ammonium hydrogen carbonate (3 × 0.2 mL, Vivaspin 500, Sartorius AG, Göttingen, Germany, 5-kDa cut-off). The protein (250 μg) was complemented with TCEP (50 mmol/L in 20 μL of 0.1 mol/L ammonium hydrogen carbonate) and incubated (60 °C, 15 min). The samples were let to cool down to room temperature (RT), alkylated with iodoacetamide (0.1 mol/L in 0.1 mol/L ammonium hydrogen carbonate, 22 μL) in the darkness (15 min, RT), the protein was digested with trypsin (25 mg/L in 50 mmol/L ammonium hydrogen carbonate, 8 μL 37°C, overnight) and the digest was stored at 80 °C. An aliquot of the sample (2 μg protein) was diluted with sample buffer (0.05% bromophenol blue, 62.5 mmol/L Tris–HCl, pH 6.8, 20% glycerol, 2% SDS, 5% βmercaptoethanol) at least two-fold and heated at 95 °C for 5 min. The sample was separated by sodium dodecylsulfate polyacrylamide gel electrophoresis[27] (SDS-PAGE, T = 12%, C = 2.65%) and stained with colloidal Coomassie Brilliant Blue G 250.[28] The digests were considered to be efficient when the HSA band was not detectable, which indicated efficiency better than 99%. The digest was diluted with aqueous acetonitrile (3% v/v) containing formic acid (0.1% v/v, eluent A) to a final volume of 1 mL and desalted by reversed phase-solid phase extraction (RP-SPE, Discovery DSC-18, 1 mL cartridge). The stationary phase was washed with eluent A (2 mL) before the peptides were eluted with aqueous acetonitrile (80% v/v) containing formic (0.1% v/v) acid (1 mL, eluent B). The eluates were dried under vacuum and stored at 20 °C prior to analysis.

Mass spectrometry

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Results Peptide synthesis The synthesis of the model sequence H-AGSAXASGFA-NH2 (1X) and the reversed one H-AFGSAXASGA-NH2 (2X), where X corresponded to arginine, Glarg, CMA, MG-H1, MG-H2, MG-H3 or CEA residues (Table 1, Protocols S1 – S6) resulted with high purities (≥95%) and yields of 26 – 28% for Glarg and 1 – 8% for MG-H containing species. CMA and CEA peptides were analyzed without any further purification. Additionally, the peptides H-ATLXETYG-OH (3X), H-LSQXFPKAE-OH (4X), H-VSDXVT-OH (5X) and H-XYKAAFTE-OH (6X), where X corresponded to Glarg, MG-H1 and MG-H2 were synthesized achieving purities of at least 95% for Glarg and MG-H2 modified peptides and of more than 90% for MG-H1 modified peptides, and yields of 10 – 31% and 1 – 18% for Glarg and MG-H containing sequences, respectively. Tandem mass spectrometry The mass spectra of peptides 1 and 2 acquired by ESI-QqTOF-MS were represented mostly by peaks of the [M + H]+ ions at m/z 893.4 in the corresponding TOF-scans. The tandem mass spectra of this m/z were dominated by b-fragment ion series which in both cases were complete (Figs. 2A and S-1A, Table S-5). Though the y-series were less intense, they were also complete, whereby the y5 – y9-ions demonstrated the highest intensity indicating the arginine residue as the preferable charge localization site. Thus, series of internal fragments containing the arginine residue on the N-terminus were also observed in the spectrum (Table S-5). The higher abundance of b-ions compared to their y-counterparts can be explained by the absence of a C-terminal residue capable to localize the charge efficiently. The low mass region of the MS/MS spectra revealed a signal at m/z 112.1 (Figs. 2A and S-1A) corresponding to an arginine immonium ion-related product of an intra-molecular SN-reaction (Im-NH3, Fig. 3A),[29] that is an indicator for arginine-containing peptides.[30] The presence of the hydroimidazolone moiety (characteristic for the most abundant AGEs – Glarg and MG-H[6]) in the arginyl side chain of the model peptides induced significant changes in fragmentation patterns. Thus, the MS/MS spectrum of the m/z 933.5 corresponding to 1Glarg dominated with the b6 fragment containing the C-terminally localized hydroimidazolone moiety, which induces an N-terminal bond breakage (Fig. 2B). While in the MS/MS spectrum of 1Glarg (Fig. 2B) the relative intensities of b5 – b10 ions were higher than those of b2 – b4, in the MS/MS spectra of m/z 893.4 (1) the b6 – b9 fragments were relatively weak (Fig. 2 A). It is

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Samples were reconstituted in aqueous acetonitrile (80% v/v) containing formic acid (0.1% v/v) and analyzed on a QSTAR Pulsar I ESI-QqTOF-MS equipped with a Turbo ion spray source (AB Sciex, Darmstadt, Germany) at a flow rate of 10 μL/min. Mass spectra and tandem mass spectra (MS/MS) were acquired and processed with Analyst QS software (version 1.1). The TOF analyzer was calibrated using a mixture of glycine, sucrose and maltoheptaose (0.1 μmol/L) in MS mode and with y1 and y11 ions of Glu-1fibrinopeptide B in MS/MS mode. All instrumental parameters are provided in Tables S-1 and S-2. Product and precursor ion scans were recorded on a QqLIT-MS (operating in QqQ mode) equipped with an ESI-Turbo V™ source (4000 Q TRAP®, AB Sciex, Darmstadt, Germany) and controlled by Analyst 1.6 software. The samples were infused in aqueous acetonitrile (80% v/v) at a flow rate of 10 μL/min. Precursor ion scan experiments were optimized for characteristic reporter ions with m/z 152.1 (Glarg) and m/z 166.1 (MG-H) using a scan rate of 0.5 μ/s (dwell time: 200 ms) from m/z 200 to 1000 with a total analysis time of 27 min. All instrumental parameters are provided in the supporting information (Tables S-1 and S-3). The HSA tryptic digest (70 ng) was loaded on a nanoAcquity UPLC™ Symmetry trap column (C18-phase, internal diameter 180 μm, length 20 mm, particle size 5 μm) at a flow rate of 5 μL/min for 5 min and then separated on a nanoAcquity UPLC™ BEH130 column (C18-phase, internal diameter 100 μm, length 100 mm, particle size 1.7 μm) using a Waters nanoACQUITY UPLC System (10 μL injection volume, full loop injection) controlled by MassLyinx X.4.1 software (Waters GmbH, Eschborn, Germany). Eluents A and B were water and acetonitrile, respectively, both containing 0.1% formic acid. Elution was performed with two

consecutive linear gradients from 3 to 50% eluent B in 45 min and to 85% eluent B in 2 min (column temperature 30°C, flow rate 0.4 μL/min). The column was connected via a PicoTip on-line nano-ESI emitter (standard coating) (outer diameter 360/20 μm, tip internal diameter 10 μm; New Objective, Berlin, Germany) to an LTQ Orbitrap XL ETD mass spectrometer equipped with a nanoESI source and controlled by Xcalibur 2.0.7 software (Thermo Fisher Scientific, Bremen, Germany). The analyses relied on DDA comprising a survey Orbitrap-scan followed by linear ion trap (LIT) MS/MS for the six most intense signals with a charge state from 2 to 5. The m/z values corresponding to AGE-modified peptides were used for an inclusion list (parent mass ± 0.4 m/z-units). The detailed settings of ion source, mass analyzer, DDA and database search are provided in the supporting information (Table S-4).

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Figure 2. Tandem mass spectra of m/z 893.5 (A), 933.5 (B), 947.5 (C) and 951.7 (D) corresponding to [M + H] ions of peptide H-AGSAXASGFA-NH2 containing arginine (1), Glarg (1Glarg), MG-H2 (1MG-H2) and CMA (1CMA) residue in position 5 (denoted as X). The spectra were acquired using an ESI-QqTOF instrument operating in positive ion mode and equipped with CID functionality with N2 as collision gas. The collision potential was set to 60 V (A–C) or 70 V (D). For each spectrum 30 scans in multi-channel acquisition (MCA) mode were acquired.

Figure 3. The proposed structures of arginine (A), Glarg (B), MG-H2 (C), Glarg (D), CMA (D) and CEA (E) reporter ions observed in the tandem mass spectra of the corresponding AGE-modified peptides at m/z 112.09, 152.08, 166.10, 170.09 and 184.11, respectively.

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important to note, that no neutral losses from the precursor, b and y fragment ions, corresponding to the modification of the arginine side chain were observed. Thus, all hydroimidazolone-containing fragment ions demonstrated the mass increment of 40 Da characteristic for Glarg. This observation was confirmed with peptide 2Glarg (Fig. S-1B). Remarkably, the same mass increment was observed for the Im-NH3 ion (Fig. 3B). This signal (m/z 152.1) displayed much higher relative intensity in comparison to m/z 112.1 in the spectrum of the unmodified peptide, though the same collision conditions were applied (Figs. 2A, B and S-1A, B).

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Further exploration of the MS/MS spectrum of m/z 933.5 corresponding to 1Glarg revealed multiple internal fragment ions containing the hydroimidozolone moiety in the N-terminal position (Fig. S-2A and Table S-5). The relative intensity of these signals (compared to the b- and y-series) was always dependent on the used CID conditions and increased with increasing collision potential (data not shown). These series of internal fragments were accompanied by corresponding ammonia neutral losses of essentially lower intensity (Table S-5). The methyl substitution in the fifth position of the imidazole ring resulted in shift of 14 Da within the whole fragmentation pattern observed in the MS/MS spectrum of m/z 933.5 (Glargcontaining peptides) without essential changes in the signal intensity profile itself (1MG-H2, Fig. 2C). The fragments containing MG-H moiety displayed a characteristic mass increment of 54 Da relative to those of unmodified peptide and did not show any neutral losses. The pattern of internal fragments resembled the one previously described for 1Glarg (Fig. S-2 and Table S-6). The same fragmentation behavior was observed for the reversed sequence 1MG-H2 (Fig. S-2B) and two other peptide-bound methylglyoxal-derived hydroimidazolones 1MG-H1, 1MG-H3, 2MG-H1 and 2MG-H3 (Fig. S-3A,B and S-4A,B). Moreover, a MG-H-related Im-NH3 signal at m/z 166.1 was observed in the low-mass region of the MS/MS spectra of all MG-H-containing peptides (Figs. 3C, S-1C, S-3A,B and S-4A,B). However, under the same collision conditions its relative intensity was lower in comparison to the signal at m/z 152.1 in the MS/MS spectra of the Glarg-containing peptides (Fig. 2 and S-1). In contrast to the described hydroimidazolone-containing peptides, the aliphatic arginine modifications (CMA and CEA) did not

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Tandem mass spectrometry for AGE detection display characteristic internal and immonium ion-related signals. Indeed, the MS/MS spectra of m/z 951.6, (1CMA), were characterized with much more complex fragmentation patterns (Fig. 2D). In this case, both b- and y-series, as well as the related internal fragment signals, displayed relatively low intensity. Moreover, these fragments were much less intense, than the corresponding neutral losses of carboxymethyl moiety, ammonia and their combination (-58, -17 and -75 u, respectively), that might indicate simultaneous fragmentation of the peptide backbone and the side chain of the CMA residue. This tendency was even more pronounced in the spectrum of m/z 965.4 (1CEA, Fig. S-3C). Moreover, internal fragments and immonium ion-related species (Figs. 3D and E) were completely absent in the spectrum. The obtained fragmentation patterns were also observed in the MS/MS spectra of the peptides with reversed sequences 2CMA and 2CEA (Figs. S-1D and S-4C). In order to evaluate the effect of the selected mass analyzer on the peptide fragmentation patterns, the MS/MS spectra of m/z 933.8 and 947.7 (1Glarg and 2MG-H2, respectively) were compared with those acquired with a hybrid quadrupole-linear ion trap (QqLIT) mass spectrometer operating in both triple quadrupole (QqQ) and QqLIT mode. All spectra displayed highly similar fragmentation patterns characterized by very intense signals at m/z 152.1 and 166.1 (Figs. 2, S-1 and S-5). These signals were also intense in the ESI-QqTOF-MS/MS spectra of 12 synthetic AGE-peptides representing human serum albumin (HSA) sequence and three different hydroimidazolones (Table 1) and did not interfere with the theoretically calculated y1, a2, b2 or internal fragment ions.

Precursor ion scanning experiments with MG-H- and Glarg-modified peptides The Im-NH3 signals at m/z 152.1 and 166.1 were intense at higher collision potentials and therefore could be considered as prospective modification-specific reporter ions for precursor ion scanning. As precursor ion scanning with a QqQ-MS might provide superior sensitivity among the compared techniques,[31] the optimization of the mass analyzer parameters was performed with peptides 1Glarg and 2MG-H2 in a QqQ mode of the hybrid QqLIT instrument. Thereby, we have focused on [M + 2H]2+ ions as only multi-charged species in the studied mass range (m/z 467.3 and 474.3 for 1Glarg and 2MG-H2, respectively, Table 1), because under ESI conditions only multiplecharged ions are relevant for proteomic MS/MS experiments.[32] For the both m/z values, in-source fragmentation was negligible at declustering potentials (DPs) lower than 60 V. The optimized collision potentials for 1Glarg and 2MG-H were 54 and 51 V, respectively. Remarkably, under these conditions [M + H]+ ions were discriminated, as their DP optimum lied above 200 V (Table S-7). As the quadrupole scan rate and dwell time directly influence the sensitivity,[33] optimization of these parameters was also performed (Table S-7). Thereby, high accuracy was achieved by setting the Q1 scanning step size to 0.1 Da for the m/z range 200 – 1000, while the maximal signal intensity for both peptides was achieved at the highest dwell time (200 ms). Hence, the scanning settings for mass calibration were adjusted correspondently. Application of the optimized parameters resulted in the total precursor ion scan duration

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Figure 4. Precursor ion scans of m/z 152.1 (A) and 166.1 (C) acquired with 250 μg/mL in vitro glycated and tryptic digested Human serum albumin and product ion scan of Glarg-modified peptide LSQ-RGlarg-FPK (B) and MG-H modified peptide LSQ-RMG-H-FPK (D). Precursor ion scans were acquired from samples dissolved in 80% aq. acetonitrile/0.1% formic acid and infused with 5 μL/min in a QqLIT mass spectrometer. Product ion scans were acquired in a DDA experiment by analyzing 1 pmol sample with a nanoUPLC-ESI-Orbitrap LTQ system.

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of 27 min. The scan duration could be, however, further reduced to 3 min with approximately 10-fold sensitivity loss (data not shown). The optimized precursor ion scanning methods were validated in terms of their sensitivity. For this, dilution series of peptides 1Glarg and 2 MG-H2 (10 pmol/L–10 nmol/L, 10-fold increment) were infused in the QqLIT mass spectrometer at 5 μL/min flow rate. The instrument limit of detection (LODi), defined as the minimal analyte concentration yielding a significant three-fold excess of signal intensity relative to the noise value,[34] was 0.1 nmol/L for both AGE-peptides (Fig. S-6A and B). For determination of the method LODs (LODm), the same peptides were serially diluted with 10 nmol/L HSA tryptic digest in 60% acetonitrile in 0.1% aq. formic acid as described above. The LODm was achieved at 0.1 and 0.5 nmol/L for 1Glarg and 2 MG-H2, respectively (Fig. S-6 C and D). Analysis of in vitro glycated HSA and plasma of diabetic patients by Glarg- and MG-H-specific precursor ion scanning The optimized method was applied to characterization of argininederived protein advanced glycation patterns. First, a tryptic digest obtained from heavily glycated HSA was scanned for m/z 152.1 and 166.1. Both reporters yielded rich patterns of intense signals in the m/z range 300 – 600 (Fig. 4A and C). To reveal the sequences underlying these signals, several consecutive RP-nanoUPLCOrbitrap-LIT-MS experiments were performed (Fig. 5). For this, the m/z values corresponding to the 60 most intense signals in precursor ion scans (Fig. 4A and C) were used to create inclusion (parent mass) lists for corresponding LC-MS/MS (DDA) experiments, comprising survey Orbitrap-MS and multiple dependent LIT-MS/MS scans (up to six per one survey scan). Following database search and manual confirmation of sequence annotation, all unassigned m/z were recalculated to their triply- and doubly-charged counterparts (with assumption that the signals in precursor ion scan corresponded to [M + 4H]4+ and [M + 3H]3+ ions, respectively), as well as related sodium and potassium adducts. Based on this data, new inclusion lists were generated and additional nanoUPLC-LTQOrbitrap-MS DDA-experiments were performed (Fig. 5). To identify

the rest of the signals, an Orbitrap-MS scan with a resolution of 100 000 was acquired and all exact masses matching unassigned signals in precursor ion scan were included in the parent mass lists of corresponding DDA-methods with mass tolerance of 10 ppm. Thereby, information about the low-abundant AGE-peptides could be retrieved. The described strategy resulted in identification of 2 Glarg- and 4 MG-H-containing peptides representing five arginine modification sites-R98, R186, R209, R222 and R472 (Table 2). Additionally, three species with side chain mass shifts of 58 and 72 m/z characteristic for dihydroimidazolidine (DH-I) intermediates of Glarg- and MG-H, respectively, were observed (Fig. S-8).[35,36] Remarkably, their sequences resembled those of Glarg- and MG-H-containing peptides (Table 2). All these modifications were identified in the first targeted LC-MS/MS (DDA). Thus, only one DDA run was sufficient for comprehensive AGE profiling. Though multiple signals present in the precursor ion scans were confirmed as AGE-modified by the subsequent LC-MS/MS (DDA) experiments, the most intense ones could not be assigned to the HSA structure (independently from the presence of AGE moiety). However, the corresponding LIT-MS/MS spectra obtained in the targeted DDA experiments (Fig. 5) demonstrated modification-specific mass shifts (Fig. S-7), confirming the high specificity of the scan. Remarkably, most of the peptides which were not assigned to the HSA sequence by the database search comprised HSA-related amino acid moieties and were often annotated with high confidence. In the next step, we acquired precursor ion scans (m/z 152.1 and 166.1) with blood plasma protein digests, obtained from three individual type 2 diabetes mellitus (T2DM) patients. Taking into account the results obtained with glycated HSA, a simplified analysis scheme, comprising only one LC-MS/MS experiment, was applied. In this context, plasma digests were scanned for m/z 152.1 and 166.1 that resulted in rich patterns of signals (Fig. 6A and B, respectively). The 150 most intense of them were selected for targeted DDA-based LC-MS/MS experiments, performed as described above. Tandem mass spectra were processed with the Sequest search engine against a human plasma protein database created from the human plasma PeptideAtlas (2012)[37] using alkylation (Cys), oxidation (Met), glycation (i.e. Amadori product formed at Lys) and AGEs (Glarg, GD-HI, MG-H and MGD-HI for Arg, as well as CM, CE for both Arg and Lys) as variable modifications. After application of search filters (post-translational modifications, rank 1, Xcorr ≥ 1.5, not more than two glycation/AGE-derived modifications in sequence and identification in all three patients), 44 peptides representing 42 proteins were annotated (Table 3). Thereby, the sequences of low-confident peptides (Xcorr ≤ 2.20 for z = 2, ≤3.75 for z = 3 and all quasi-molecular ions with z > 3) were confirmed by manual interpretation. Arginine modifications were mostly represented by Glarg (22 peptides/24 sites), followed with GD-HI (12 peptides/15 sites), MG-H (8 peptides/8 sites) and MGD-HI (6 peptides/6 sites). Additionally, further 207 AGE-containing peptides were annotated by application of the same search filters in one and two samples (Table S-8).

Discussion AGE-specific MS/MS fragmentation patterns

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Figure 5. The workflow for identification of hydroimidazolone-containing peptides in tryptic digests.

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When bottom-up proteomic approach is used to study posttranslational modifications, both annotation of modified proteins by a database search and localization of modified residues therein,

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Table 2. Glarg-, MG-H- and dihydroxyimidazolidine-modified peptides detected in tryptic digest of in vitro glycated HSA by modification-specific precursor ion scanning and identified in subsequent RP-nanoUPLC-ESI-Orbitrap-LTQ-MS-based DDA experiments No

A1 A2 A3 A4 A5 A6 A7 A8 A9

Precursor ion scan

nanoLC-Orbitrap-LIT-MS DDA

m/z

Reporter ion (m/z)

tR (min)

m/z

353.0 377.1 574.0 465.4 474.7 454.9 458.4 567.0 467.5

166.1 166.1 166.1 166.1 166.1 166.1 152.1 152.1 166.1

17.26 19.02 19.28 19.52 20.50 21.34 23.52 23.81 25.17

352.86 376.85 573.78 465.26 474.27 454.73 458.25 566.59 467.26

+

m/z [M + H] 1056.56 1128.55 1146.56 929.52 947.52 908.46 915.50 1697.76 933.51

XCorr

Probability

Sequence annotation

Modification site

1.37 2.10 1.87 1.37 1.90 2.55 2.08 2.66 2.25

1.00 1.00 1.00 1.00 1.00 1.12 1.00 1.00 1.00

TPVSD-[MG-H]-VTK LDEL-[MG-H]-DEGK LDEL-[DH-I]-DEGK a LSQ-[MG-H]-FPK LSQ-[DH-I]-FPK a FGE-[MG-H]-AFK LSQ-[Glarg]-FPK QEPE-[Glarg]-NECFLQHK FGE-[DH-I]-AFK a

R472 R186 R186 R222 R222 R209 R222 R98 R209

a

DH-I was found when searching in database for CEA as both give the same mass shift.

Figure 6. Precursor ion scans for m/z 152.1 (A) and 166.1 (B) acquired with 100 μg/mL tryptic digested plasma proteins of a diabetic patient. Precursor ion scans were acquired from samples dissolved in 80% aq. acetonitrile/0.1% formic acid and infused with 5 μL/min in a QqLIT mass spectrometer.

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arginyl side chain would essentially change proton affinity of this residue, and, hence, its ability to localize charge and drive fragmentation. Fortunately, our results confirmed stability of Glarg- and MG-H under CID conditions and applicability of specific mass increments of 40 and 54 Da, respectively, for the sequencing of corresponding AGE peptides (Fig. 2B and C).[22,42] In contrast, CMA and CEA showed only limited stability under CID conditions (Fig. 2D and S-1C). Hence, the increments of 58 and 72 Da do not unambiguously indicate these modifications, though they were observed in tandem mass spectra of tryptic protein digests.[22] Obviously, additional MS/MS experiments are required in such cases. Cotham, Brock et al. assumed, that fragment ions with these mass shifts originate from dihydroxyimidazolidine intermediates of Glarg[22] and MG-H[43] formation (isobaric to CMA and CEA, respectively).[36,44] Indeed, the MS/MS fragmentation patterns of dihydroxyimidazolidine-modified peptides represent these mass increments, but also intense patterns of internal fragment signals characteristic for Glarg and MG-H and Im-NH3 ion peaks at m/z 152.1 and 166.1.[45] These internal fragment ion series were rather intense in the MS/MS spectra of hydroimidazolone-containing peptides, probably due to enhanced charge localization on the modified arginyl

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typically rely on characteristic modification-specific mass increments. In this context, a special emphasis should be given to the stability of modifications as simultaneous fragmentation of the peptide backbone and the side chain moiety very often make the spectrum difficult for interpretation and increase the probability of false-positive results.[38] Though database search based on AGE-specific mass increments was already used in proteomic studies,[39] the information about the influence of the corresponding AGE structures on the peptide fragmentation patterns is not available so far. To address this question we acquired tandem mass spectra of AGSAXASGFA-NH2 (1X) and its reversed sequence H-AFGSAXASGA-NH2 (2X) containing specific AGEs (X) in a unique arginine position. This approach was expected to deliver complementary structure information (i.e. double b- and y-ion series) and thereby provide detection of all modification-related fragment ions. Amino acid residues, known to essentially influence fragmentation patterns of peptides and thereby interfere with modification-specific signals (e.g. internal proline or histidine[40] and C-terminal lysine or arginine[41] residues), were avoided. As peptides 1 and 2 did not produce any unexpected sequencerelated signals, they would be a convenient model to characterize possible side chain fragmentation pathways. We also assumed that the presence of a new structure moiety in the

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Table 3. Glarg-, GD-HI-, MG-H- and MGD-HI-containing peptides detected in three T2DM blood plasma protein tryptic digests by modification-specific precursor ion scanning (m/z 152.1 and 166.1) and identified in subsequent single targeted RP-nanoUPLC-ESI-Orbitrap-LTQ-MS-based DDA experiments Label P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 P39 P40 P41 P42 P43 P44

Sequence IKCMLWEVYGER-[Glarg] AMRNKLEGEI-[Glarg]-R C*QVCM-[Glarg]-RT FC*FSN-[Glarg]-MOxSTMTPK EGVFD-[Glarg]-MKVALDK DGEKCES-[GD-HI]-VKVK [MG-H]-GRPAATEVKCE [Glarg]-PSRWPAMKCM [GD-HI]-KAmadoriANFKDQ KCM-[GD-HI]-EEEEKAQQVAR [Glarg]-QLEEEVKAK EKCMRE-[MGD-HI]-SR [Glarg]-HTKAmadoriEK YID-[MG-DHI/RCE]-IHIFF-[Glarg] YID-[MG-DHI/RCE]-IHIFF-[Glarg] NTLYLQMOxN-[MG-H]-LR LS-[Glarg]-IIT-[GD-HI]-IQAQSR VK-[MGD-HI]-QNSLKAmadoriEWR I-[MGD-HI]-DAL-[MG-H]-SILTISR VLDHF-[Glarg]-[Glarg]-K [GD-HI]-TGC*IGAK V-[GD-HI]-GGVPLVK MOxNK-[MG-H]-ILGGR VTVMASLR-[Glarg]-PKCMR QLVNEDL-[MGD-HI]-K LPSF-[GD-HI]-[GD-HI]-YVEKQASEK YHLGMYH-[Glarg]-RINRVTDR [Glarg]-TASPPPPPK D-[Glarg]-RGSLEKAmadoriSQGDK [Glarg]-[Glarg]-GFCOxFITFKEEEPVK [MG-DHI/RCE]-ITA-[Glarg]-KDNILR [MG-H]-ITARKCMDNILR GVVMLN-[Glarg]-VMEKGSLKCMCAQYWOxPQK [GD-HI]-GQSP-[GD-HI]-LLIR MOxEQLLRAEL-[GD-HI]-TATLR LLEKCELSLENCGLT-[GD-HI]-R FGCOxIS-[MG-H]-KCMCPAIR [Glarg]-IVACOxASYKCEPSR RQNGI-[MGD-HI]-EAVSLL-[MG-H]-R EVSVM-[Glarg]-DEDKLLR FQQAVDAVEEFL-[GD-HI]-RAK [Glarg]-DKCEIIVNDRQLACOxAR AQLSVKN-[G-DHI/RCM]RQ-[MG-H]-PSR FG-[GD-HI]-Q-[GD-HI]-KIGGR GTLDPVEKAmadoriAL-[Glarg]-DAKLDK

a

tR (min) 17.03 17.97 18.73 19.30 20.22 21.71 21.85 22.04 22.26 23.41 23.52 23.58 23.79 23.81 23.99 24.04 25.86 26.03 26.29 26.64 26.85 27.36 27.63 27.69 28.06 28.75 28.83 28.87 29.12 29.80 30.39 30.78 31.24 32.51 32.61 32.66 32.91 32.92 34.94 36.50 36.84 37.34 38.11 38.16 38.66

m/z

z

XCorr

Protein

387.45 504.94 547.24 554.91 774.40 638.35 605.83 409.54 409.54 572.96 423.90 364.19 500.75 497.93 373.70 497.92 547.32 420.23 547.32 575.80 460.73 491.80 557.81 504.62 593.83 652.00 709.70 544.29 559.94 694.67 489.95 489.95 695.59 437.92 469.51 625.68 493.58 493.58 598.67 543.95 655.67 475.50 603.34 645.86 691.04

4 3 2 3 2 2 2 3 3 3 3 3 2 3 4 3 3 4 3 2 2 2 2 3 2 3 3 2 3 3 3 3 4 3 4 3 3 3 3 3 3 4 3 2 3

1.83 1.99 1.62 2.32 2.25 1.51 1.55 1.93 2.08 2.76 1.85 1.75 1.50 2.04 2.14 2.43 2.55 1.56 2.19 1.66 1.64 1.64 1.72 2.50 1.79 2.08 2.45 1.50 1.96 1.85 2.81 2.17 2.63 2.98 1.91 2.53 2.16 2.29 1.94 2.82 2.21 3.02 1.52 1.83 2.22

Pre-mRNA-processing factor 17 Angiomotin-like protein 1 Collagen alpha-1(IV) chain Transmembrane emp24 domain-containing protein 2 cDNA FLJ12864 fis, highly similar to Alpha-catulin Heat shock protein 105 kDa PC4 and SFRS1-interacting protein Plexin domain-containing protein 2 Enoyl-CoA hydratase, mitochondrial Hematopoietic line age cell-specific protein Myosin-7 Serine/arginine repetitive matrix protein 1 Desmocollin-2 Pre-mRNA-processing-splicing factor 8 Pre-mRNA-processing-splicing factor 8 Anti-HER3 scFv Myosin-7 Short transient receptor potential channel 1 Methionine--tRNA ligase, cytoplasmic Structural maintenance of chromosomes protein 3 60S ribosomal protein L11 Eukaryotic translation initiation factor 3 subunit C Atrial natriuretic peptide-converting enzyme DNA-dependent protein kinase catalytic subunit ATPase family AAA domain-containing protein 3C Origin recognition complex subunit 3 2-oxoglutarate dehydrogenase, mitochondrial Serine/arginine repetitive matrix protein 1 Putative RNA-binding protein 15 Heterogeneous nuclear ribonucleoprotein D0 Spectrin beta chain, erythrocytic Spectrin beta chain, erythrocytic Tyrosine-protein phosphatase non-receptor type 1 Ig kappa chain V-III region IARC/BL41 Fructosamine-3-kinase Inter-alpha-trypsin inhibitor heavy chain H5 Stanniocalcin-2 Complement C5 Plakophilin-1 Kinesin-like protein KIF20B Putative oxidoreductase GLYR1 tRNA-splicing ligase RtcB homolog Neurabin-1 Immunoglobulin superfamily member 10 Heat shock cognate 71-kDa protein

The blood samples were obtained from three T2DM individuals; C*, carbamidomethyl cysteine; Cox—oxidized cysteine, sulfenic acid; MOx—oxidized a methionine, methionine sulfoxide; the acquisition times of corresponding LIT-MS/MS scans are given.

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residue. Their formation may be described by the cyclization mechanism proposed by Bythell et al.,[46] i.e. nucleophilic attack of an hydroimidazolone electron pair on variable peptide backbone carbonyl carbon atoms C-terminally from the modification site (Fig. S-8). As the same mechanism underlines the formation of arginine-derived immonium ion-related species[29] (Im-NH3, Fig. 3A), the MG-H- and Glarg-derived signals of this type (m/z 152.1

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and 166.1, Fig. 3B and C) were intense in the spectra of the corresponding AGE peptides (Figs. 2B, C and S-2B, C) as well. These species together with ammonia neutral loss patterns from modification-specific internal fragments provide deeper insight in the above mentioned cyclization mechanism.[46] Indeed, imine nitrogen of hydroimidazolone moiety is the most probable donor of electron pair in the intermolecular SN-reaction, as the resulting

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Tandem mass spectrometry for AGE detection cyclic structure hinders further ammonia cleavage (Fig. S-8B). In contrast, when guanidine group of arginine residue is involved in cyclization, further cleavage of amino group is highly probable (Fig. S-8A). It also explains the relatively low intensity of immonium ion related signals in the spectra of CMA-containing peptides (Figs. 2D and S-2D). In summary, all hydroimidazolone-modified peptides demonstrated several common features in their fragmentation patterns: (i) characteristic modification specific mass increments of 40 or 54 Da, (ii) high intensity of the modification-containing fragment ions, (iii) presence of modification-specific signals at 152.1 and 166.1 corresponding to Im-NH3 ions and (iv) very intense signals of internal fragment ions. Thus, the use of several features of MS/MS spectra in addition to mass increment-based database search could increase the reliability of peptide annotation and identification of modification sites. Precursor ion scanning

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Detection of AGE-modified residues in human serum albumin (HSA) The applicability of our method for the analysis of glycated proteins was verified in the model of heavily glycated HSA. As this polypeptide comprises approximately 50% of plasma protein content and has a relatively long half-life (21 days), it might be diagnostically relevant and could serve as a convenient model for further extension of our approach to in vivo plasma samples. Precursor ion scanning revealed prevalence of prospectively highly-charged quasimolecular ion signals, that might indicate multiple missed cleavage sites, related to side chain modifications.[49] Remarkably, the identity of five modification sites (Table 2) was confirmed already in the first targeted DDA experiment (Fig. 5). It means that in most cases one infusion-based precursor ion scan and one LC-MS/MS experiment (taking together approximately 1.5 h) are sufficient for the characterisation of a specific AGE in tryptic digest. Interestingly, not only Glarg and MG-H, but also their D-HI precursors were detected. Simultaneous observation of peptides containing methylglyoxal-derived hydroimidazolones and dihydroxyimidazolidines was described in in vitro glycated plasma proteins with methylglyoxal.[50] Thus, detection of two sequential steps of MG-H and Glarg formation in combination with MS/MSbased sequence assignment provided reliable identification of correspondingly modified peptides. The modification sites detected here (R186, R209, R222 and R472, Table 2) differ from the patterns described in literature. Altogether four MG-H modification sites, namely R10, R98, R218 and R428, were annotated in HSA minimally glycated in vitro by MALDI-TOFMS.[51] Probably, due to severe conditions, this pattern was distinctly different from that produced by minimal methylglyoxal treatment (R114, R186, R218, R410 and R428).[52] Under the conditions applied here, relatively reactive arginine residues could be involved in further oxidative and cross-linking reactions. Alternatively, MG-H can undergo reaction with another methylglyoxal molecule (product of glucose autoxidation) resulting in the formation of argpyrimidine.[36] Therefore, here detected modifications may represent less reactive arginine residues. Similarly, two of the Glarg modification sites (R98 and R222, Table 2) detected here do not match with the results of two published studies with minimally in vitro glycated HSA. The corresponding arginine residues described by the authors (R251, R428[51] and R160, R472[53]) were probably too reactive to be detected in our model of heavily glycated HSA. However, it is necessary to note, that the structure of the hydroimidazolone- and dihydroxyimidazolidine-modified peptides detected here was proven by MS/MS, that to the best of our knowledge, was done for the first time in the models of in vitro HSA glycation. Unexpectedly, many signals observed in precursor ion scans could not be assigned to HSA sequence in targeted nanoUPLCMS/MS experiments. We assumed that these sequences corresponded to the species containing multiple post-translational modifications, most probably, cross-links or side chain oxidation (e.g. cysteine, tyrosine, tryptophan residues) by their nature. Indeed, intensive formation of intermolecular cross-links was previously

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The modification-specific character of the signals at m/z 152.1 and 166.1 allows rapid and specific detection of the underlying hydroimidazolone modifications, (Glarg and MG-Hs, respectively) by precursor ion scanning. As these AGEs are the major clinically relevant arginine-derived glucoxidation products,[20] this approach might be a promising tool in biomarker research and related diagnostic strategies. To compare the potential of different mass spectrometers for precursor ion scanning, we performed MS/MS experiments with ESI-QqTOF and QqLIT instruments. As the latter instrument combines triple quadrupole (QqQ) and linear ion trap (LIT) technologies,[47] we were able to compare the potential of three most widely commercialized tandem-in-space mass analyzers (QqQ, as well as its hybrid versions QqTOF and QqLIT) to produce modification-specific fragments under CID-MS/MS conditions. As all three MS techniques delivered intense reporter ions, the signals at m/z 152.1 and 166.1 could be considered as universal for tandem-in-space mass spectrometers. Thus, these signals could be used for development of modification-specific precursor ion scans, applicable for a wide range of commercial instruments. Here, in order to provide higher sensitivity,[31] we used a hybrid QqLIT instrument (operating in a QqQ mode) mass spectrometer for precursor ion scanning experiments. However, one should keep in mind, that any other tandem-in-space instrument can be used for this purpose, whereas tandem-intime mass spectrometers (e.g. linear or three dimensional ion traps) have only limited application due to the low mass cutoff effect.[31] The optimized precursor ion scans proved to be highly specific not only for modification type (no cross-talk with the signals of unmodified species), but also the charge state of quasimolecular ions: singly charged ions were completely excluded at low DP values optimal for desolvatation of multiply charged species (Table S-7). This feature could be advantageous for analysis of tryptic protein digests, as these systems are represented mostly by peptides at higher charge states.[32] High sensitivity was another advantage: the achieved limits of detection (up to 0.5 nmol/L) were comparable with those obtained on the amino acid level[6] In this context, these precursor ion scanning methods are advantageous, when complex discovery experiments are to be performed. Indeed, a precursor ion scan followed by one or several nanoLC-MS runs for detection and structural assignment of AGE-peptides, respectively, may

efficiently replace or simplify time-consuming strategies, such as gas phase fractionation (GPF)[48] or 2D-chromatography. Furthermore, the combination of features such as low run time and high sensitivity makes the described method a promising potential screening tool.

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shown to accompany in vitro protein glycation reactions.[54] Under the glycation conditions used here, intensive autoxidation of both free glucose and protein-bound fructosamines (Amadori products) accompanied with enhanced production of ROS take place.[55] These agents (first of all, hydroxyl and superoxide radicals) readily oxidize the side chains of amino acid residues, leading to formation of cross-links (e.g. bityrosine or pentosidine) and protein backbone fragmentation.[56] Besides, protein-bound autoxidation products of fructosamines can easily react with unmodified lysine and arginine residues resulting in formation of various intra- and intermolecular cross-linked AGEs.[3] In both cases, worse digestibility with trypsin may be expected. This in combination with multiple modifications could interfere with the database search and thus make sequence assignment of tryptic peptides a challenging task. To assign the structure of these species, an additional separation step prior to precursor ion scanning and MSn experiments would be necessary. However, the structural characterization of these species was behind the scope of this work. Detection of AGE-modified residues in human blood plasma proteins

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Under the conditions of diabetic hyperglycemia, blood plasma proteins are subjected to high concentrations of glucose. In the presence of the trace concentrations of transition metal ions this sugar undergoes autoxidation, accompanied with formation of glyoxal and methylflyoxal, i.e. precursors of hydroimidazolonerelated AGEs.[45] However, due to low sugar concentrations and reaction temperatures (37°), these conditions can be considered as relatively mild and similar to the minimal glycation model. In this context, lower degree of protein oxidation, cross-linking and adduct formation can be expected. This would provide higher peptide/protein identification rates and lower number of unassigned sequences. Indeed, both reporter ions yielded rich patterns of signals in corresponding precursor ion scans (Fig. 6). Remarkably, the scan for the Glarg-specific fragment (m/z 152.1) resulted in higher numbers of signals (especially in lower m/z range, Fig. 6A), while the scan for the MG-H-specific one represented multiple signals with m/z > 700 (Fig. 6B). Expectedly, it was impossible to identify the corresponding modified peptides by MS/MS infusion experiments (data not shown). It can be explained not only by low abundances of AGE-containing peptides, but also essential matrix effects, typical for this kind of experiments. In contrast, LC-MS/MS analysis revealed 44 peptides, containing arginine-derived AGEs and representing 42 proteins modified in all three T2DM plasma samples (Table 3). Moreover, 207 further AGE-peptides were annotated only in one or two plasma samples (Table S-8). Surprisingly, no highly-abundant proteins were found to be glycated at arginine residues. It can be explained by relatively low protein half-lives (and, probably, Arg contents) and was in accordance with our previous observations.[57] Thereby, the largest protein group annotated in all samples (comprising 13 species) was represented by cytoskeleton molecules (Table 3). These proteins are typically present in plasma,[58] and, due to relatively long half-lives, can be glycated.[57] Regulatory proteins and enzymes (9 and 10 glycated species, respectively, Table 3), typically have wellexposed Arg residues in their sequence, and can be, therefore, AGE-modified at these sites, as was shown for lysine-derived AGEs.[57] Remarkably, among enzymes, identified as AGE-modified was fructosamine-3-kinase—the key enzyme of anti-glycation defence.[59] Thus, our result raises a question about the role of GD-HI modification of a specific protein residue. Indeed, this

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posttranslational modification might play a signalling role, or modulate enzyme activity, that must be the object of further studies.

Conclusions AGE-modified peptides are expected to be a new generation of disease-specific biomarkers. As their sequences include characteristic advanced glycation sites, these species may deliver direct information about changes in protein structure and function. This approach might provide fine monitoring of disease treatment. However, due to the low abundance of individual AGE-peptides (underlined by a large variety of possible formation pathways), their detection by untargeted LC-MS/MS (DDA) experiments is often compromised. Here we described precursor ion scanning methods for detection of AGE-peptides which provide the possibility to overcome this ‘undersampling’ effect by their combination with targeted LC-MS/MS (DDA) experiments. The identified Glarg- and MG-H-specific reporter ions (m/z 152.1 and 166.1) proved to be reliable markers of hydroimidazolone formation. Our strategy resulted in identification of nine hydroimidazolone-containing peptides representing six modification sites in heavily glycated HSA. The same strategy revealed 42 plasma proteins modified by their arginine residues and represented in blood of three T2DM individuals. Thus, our method may be applied to discovery and/or profiling experiments with human plasma with the aim to identify prospective hydroimidazolone peptide biomarkers of T2DM patients. Acknowledgements We thank Dr. Ralf Hoffmann for providing facilities, financial support and helpful discussions, as well as Prof. Matthias Blüher and M. Sc. Sanja Milkovska for providing plasma samples and language proof reading, respectively. Financial support from the ‘Deutsche Forschungsgemeinschaft’ (DFG, HO-2222/7-1 and FR-3117/2-1 the European Fund for Regional Structure Development (EFRE, European Union and Free State Saxony) and the ‘Bundesministerium für Bildung and Forschung’ (BMBF) to RH are gratefully acknowledged.

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[31]

[32] [33]

[34] [35] [36]

[37]

[38] [39]

[40] [41] [42]

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