Sensitive and Site-Specific Identification of Carboxymethylated and

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Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma Uta Graifenhagen, Viet Duc Nguyen, Johann Moschner, Athanassios Giannis, Andrej Frolov, and Ralf Hoffmann J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr500799m • Publication Date (Web): 25 Nov 2014 Downloaded from http://pubs.acs.org on December 11, 2014

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Journal of Proteome Research

Sensitive and site-specific identification of carboxymethylated and carboxyethylated peptides in tryptic digests of proteins and human plasma

Uta Greifenhagen1,2, Viet Duc Nguyen1,2, Johann Moschner3, Athanassios Giannis2,3, Andrej Frolov1,2*, and Ralf Hoffmann1,2

1

Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität

Leipzig 2

Center for Biotechnology and Biomedicine (BBZ), Universität Leipzig

3

Institute of Organic Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig

*Corresponding author Dr. Andrej Frolov Institut für Bioanalytische Chemie Biotechnologisch-Biomedizinisches Zentrum (BBZ) Deutscher Platz 5 04103 Leipzig Germany Tel.

+49 (0) 341 9731332

Fax.

+49 (0) 341 9731339

E-mail: [email protected]

Key words: AGEs; CEL; CML; glycation; precursor ion scan; tandem mass spectrometry

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Abstract Glycation refers to a non-enzymatic post-translational modification formed by the reaction of amino groups and reducing sugars. Consecutive oxidation and degradation can produce advanced glycation end-products (AGEs), such as Nε-(carboxyethyl)- (CEL)- and Nε(carboxymethyl)lysine (CML). Although CEL and CML are considered as markers of arteriosclerosis, diabetes mellitus, and ageing, the modified proteins and the exact modification sites are mostly unknown due to their low frequency and the lack of their enrichment strategies. Here we report characteristic fragmentation patterns of CML- and CEL-containing peptides and two modification-specific reporter ions for each modification (CML: m/z 142.1 and 187.1; CEL: m/z 156.1 and 201.1). The protocol allowed sensitive and selective precursor ion scans to detect the modified peptides in complex sample mixtures. The corresponding m/z-values identified eight CEL/CML-modification sites in glycated human serum albumin (HSA) by targeted nano-RPC-MS/MS. The same strategy revealed 21 CMLsites in 17 different proteins, including modified lysine residues 88 and 396 of human serum albumin, in a pooled plasma sample that was obtained from patients with type 2 diabetes mellitus.

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Introduction Glycation (or non-enzymatic glycosylation) is a common non-enzymatic protein modification in living organisms initiated by the reaction of aldoses and ketoses with amino groups.1 The resulting Schiff bases can rearrange to Amadori2 and Heyns3 compounds, respectively. Both compounds are prone to oxidative degradation and cross-linking reactions, yielding a highly heterogeneous group of advanced glycation end-products (AGEs).4 Dicarbonyl compounds, such as glyoxal, methylglyoxal, and 3-deoxyglucosone, originating mostly from oxidative degradation of sugars and lipids, are important intermediates in the formation of AGEs1. Nε(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML) are the best studied lysinederived AGEs (Figure 1A).5,6 CML- and CEL-modifications are often suggested as biomarkers, as they are linked to the pathogenesis of arteriosclerosis, diabetes mellitus, uremia, heart failure, and neurodegenerative disorders. Furthermore, both modifications are related to distinct oxidative and carbonyl stress conditions.4,6,7 Global glycation levels can be measured by enzyme-linked immunosorbent assays (ELISA)7,8 and immunoblotting,9,10 techniques that have limitations by the panel of existing crossreactivity of antibodies.11 Amino acid analysis of protein hydrolysates appears to be more universal, such as derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate followed by reversed-phase high-performance liquid chromatography (RP-HPLC),12 ionexchange chromatography combined with ninhydrin detection,13 GC-MS of trifluoroacetyl derivatives14 or LC-MS. MS techniques, which typically rely on stable isotope-labeled AGEs as internal standards, provide high sensitivity and accuracy.15,16 While all the abovementioned techniques provide the global modification degrees of a protein mixture, they cannot identify modified proteins and modification sites. This information, however, is required for a better understanding of the underlying disease mechanisms and for identifying biomarkers, and could in principle be obtained by current proteomics techniques.17 Indeed, CML- and CEL-modification sites have been identified in abundant proteins, such as in 3 Environment ACS Paragon Plus


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crystallins18 and collagens19 isolated from lens and connective tissue, respectively. In complex protein mixtures, such as plasma, AGE-modifications are typically missed due to their low frequency and the lack of specific enrichment strategies. Here we report sensitive and selective strategies to identify CML- and CEL-sites in protein digests using fragmentation patterns and reporter ions specific for modified peptides. Based on these signals, a sensitive and specific precursor ion scan followed by a targeted nano-RPCMS/MS protocol was established and validated for plasma samples. This strategy identified 21 CML- and CEL-modification sites in 17 plasma proteins with partially low abundances.

Materials and Methods Unless stated otherwise, materials were obtained from the following manufacturers: AB Sciex (Darmstadt, Germany): polypropylene solutions and ESI Tuning Mix; AppliChem GmbH (Darmstadt, Germany): tris (ultrapure); Biosolve (Valkenswaard, NL): acetonitrile (MS grade),

N,N’-dimethylformamide

(DMF, ≥ 99.8%),

piperidine

(≥ 99.5%),

and

dichloromethane (DCM, ≥ 99.9%); Carl Roth GmbH & Co KG (Karlsruhe, Germany): trifluoroacetic acid (≥ 99.9%), glacial acetic acid, methanol (≥ 99.9%), dimethyl sulfide (p.a.), α-D-glucose

monohydrate

(≥ 99.5%),

sodium

dodecylsulfate

(≥ 99.5%),

tris-(2-

carboxyethyl)phosphine hydrochloride (≥ 98%), glycerin (≥ 99.5%) and silicagel 60; Conlac GmbH (Leipzig, Germany): hexane (puriss); Iris Biotech GmbH (Marktredwitz, Germany): 9Fluorenylmethoxycarbonyl (Fmoc-) Rink Amide AM resin and Fmoc-L-Gln(Trt)-OH (peptide synthesis grade); MultiSynTech GmbH (Witten, Germany): Fmoc-L-Trp(Boc)-OH; ORPEGEN Pharma (Heidelberg, Germany): Fmoc-Nε-(allyloxycarbonyl)lysine (FmocLys(Alloc)-OH) and all other Fmoc-protected L-amino acids (peptide synthesis grade); New Objective (Berlin, Germany): PicoTip on-line nano-ESI emitter with standard coating (outer diameter 360/20 µm, tip internal diameter (ID) 10 µm; Promega GmbH (Mannheim,


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Germany): trypsin (premium grade); SERVA Electrophoresis GmbH (Heidelberg, Germany): acrylamide/bis-acrylamide solution (37.5/1, 30% (w/v), 2.6% C), ammonium persulfate (p.a.), N,N,N′,N′-tetramethylethane-1,2-diamine (research grade), Coomassie Brilliant Blue G 250 (pure), glycine (p.a.), and sucrose (> 98%); Solvadis GmbH (Frankfurt am Main, Germany): ethyl acetate (puriss); VWR International GmbH (Dresden, Germany): diethyl ether (100%) and acetonitrile (≥ 99.9%). Waters (Waters GmbH, Eschborn, Germany): nanoAcquity UPLC Symmetry trap column (C18-phase, ID 180 µm, length 2 cm, particle size 5 µm) and nanoAcquity UPLC BEH130 column (C18-phase, ID 0.1 mm, length 10 cm, particle size 1.7 µm). All other chemicals were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany); Glu-1-fibrinopeptide B (H-EGVNDNEEGFFSAR-OH) was synthesized and purified in house. Water was purified in house (resistance > 18 mΩ/cm; total organic content < 1 ppb) on a PureLab Ultra Analytic System (ELGA Lab Water, Celle, Germany). The diabetes plasma samples were obtained by pooling three individual samples obtained from diabetes type 2 patients in accordance with regulations of ethical committee of the Universität Leipzig.

Peptide synthesis Reagents and materials Peptides with sequences AGSAXASGFA-NH2 (X = Lys, CML or CEL for peptides 1, 2 or 3), ERAFXAWAV-NH2 (X = CML or CEL for peptides 4 or 5), KQTALVELVK (X = CML or CEL for peptides 6 or 7) and LAXTYETTLEK (X = CML or CEL for peptides 8 or 9) were synthesized on a Syro2000 multiple peptide synthesizer (MultiSynTech GmbH, Witten, Germany) by Fmoc/tert-butyl-chemistry using eight equivalents (eq.) of Fmoc-amino acid derivatives activated with N,N’-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) in DMF (Table 1).20 Lysine residues to be modified were incorporated as Fmoc-Lys(Alloc)OH. After coupling the N-terminal residue, the Fmoc-group was cleaved with piperidine and 5 Environment ACS Paragon Plus


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the N-terminus remained protected with the butyloxycarbonyl (Boc) group. The Alloc group was selectively cleaved with tetrakis(triphenylphosphine)palladium(0).21 The lysyl side chains were alkylated with tBu glyoxal or tBu pyruvate (40 eq., DMSO solution diluted to 61 mmol/L with DCM) and DIPEA (1.1 eq., 69 mmol/L) in the presence of NaCNBH3 (20 eq., 300 mmol/L in CH3OH, DCM 1/1) by stirring at 4 °C overnight. Tert-butyl (tBu)glyoxal and tBu pyruvate were synthesized from fumaryl chloride22 and tBu methacrylate,23 respectively, using slight modifications of the original protocol (Supporting information, Protocol S-1). The final peptide resins were washed with DCM, dried, and cleaved with TFA containing 12.5% (v/v) of a scavenger mixture (ethanedithiol, m-cresol, thioanisole, and water, 1:2:2:2 by volume). After 2 h the peptides were precipitated and washed twice with cold diethyl ether, dried, and purified on a C18 column using a linear aqueous acetonitrile gradient in the presence of 0.1% (v/v) TFA or 0.1 % (v/v) heptafluorobutyric acid (HFBA) as ion-pairing reagent (Supporting information, Protocol S-2).24,25

In vitro glycation of human serum albumin (HSA) HSA (5 mg) was dissolved in sodium phosphate buffer (0.1 mol/L, pH 7.4, 350 µL) containing α-D-glucose (0.5 mol/L) and incubated under continuous shaking (400 rpm, 50 °C) for seven days. The sample was split in 0.1 mL aliquots and stored at -20 ºC.

Trypsin digest of glycated HSA and human plasma One mg of glycated HSA or 0.5 mg of human plasma were ultra-filtrated for 30 min at 10,000 rpm against ammonium bicarbonate (0.1 mol/L; 3 x 0.2 mL, Vivaspin 500, Sartorius, Göttingen, Germany, 5 kDa cut-off). The filtrates were complemented with tris(2carboxyethyl)phosphine (TCEP, 50 mmol/L in water, 10 µL), diluted with aqueous ammonium hydrogen carbonate solution (50 mmol/L) to obtain a final volume of 100 µL, and incubated (60 °C, 15 min). The samples were cooled to room temperature (RT), alkylated 6 Environment ACS Paragon Plus

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with iodoacetamide (0.1 mol/L, 11 µL) in the darkness (15 min, RT), and digested with trypsin (25 mg/L in 50 mmol/L ammonium hydrogen carbonate, 1 mL, 37 °C, over night). Next morning the digest was frozen and stored at -80 °C. An aliquot (2 µg protein) of the sample was diluted with sample buffer (0.05% bromophenol blue, 62.5 mmol/L Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol) at least twofold and heated to 95 °C for 5 min. The sample was separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, T=12%, C=2.65%)26 and stained with colloidal Coomassie Brilliant Blue G 250.27 The digestion steps were considered efficient when the HSA band was no longer detectable, indicating a digest efficiency better than 95%. One and a half mL digest was mixed with 45 µL acetonitrile and 6 µL formic acid, and loaded on a Discovery DSC-18 1 mL reversed phase solid phase extraction (RP-SPE) cartridge (Supelco, Sigma Aldrich GmbH, Taufkirchen, Germany). After washing with 2 mL of 3% (v/v) aq. acetonitrile containing 0.1% (v/v) formic acid, retained tryptic peptides were eluted with 1 mL 80% (v/v) aq. acetonitrile in 0.1% (v/v) formic acid.

Mass spectrometry Samples were analyzed in positive ion mode on an ESI-QqLIT-MS instrument (4000 QTrap™) equipped with a Turbo VTM ion source and controlled by the Analyst 1.6 software (AB Sciex, Darmstadt, Germany). The quadrupole was tuned and calibrated with polypropylene glycol for unit and high resolutions defined as peak widths of 0.7 ± 0.1 or 0.5 ± 0.1 m/z-units, respectively. Samples were dissolved in aqueous acetonitrile (60%, v/v) containing formic acid (0.1%, v/v) and infused at a flow rate of 5 µL/min (PHD 2600 syringe pump, Harvard Apparatus, Holliston, MA, USA). Precursor ion scans were optimized for characteristic reporter ions at m/z 142.1 and 187.1 (CML) or m/z 156.1 and 201.1 (CEL) in a mass range from m/z 300 to 900 at a Q1 scan rate of 0.5 unit/s and a step size of 0.1 m/z-units. Further parameters are provided in supporting information (Table S-1). 7 Environment ACS Paragon Plus


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The tryptic digest of HSA (70 ng) was loaded on the trap column at a flow rate of 5 µL/min for 5 min and then separated on the BEH130 column using a nano-ACQUITY UPLC System (10 µL injection volume, full loop injection) controlled by the MassLynx X.4.1 software (Waters GmbH). The UPLC was connected via a PicoTip on-line nano-ESI emitter to an LTQ Orbitrap XL ETD MS equipped with a nano-ESI source and controlled by Xcalibur 2.0.7 software (Thermo Fisher Scientific, Bremen, Germany). Eluents A and B were water and acetonitrile, respectively, both containing 0.1% (v/v) 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). Analyses relied on a survey Orbitrap-scan followed by CID fragmentation in the LIT in data-dependent acquisition (DDA) mode for the six most intense signals with charge states from 2 to 5. M/z-values corresponding to prospective AGE-modified peptides were used for an inclusion list (parent mass ± 0.4 m/z-units). More details of the protocol are provided in supporting information (Table S-2). Tandem mass spectra were processed with the Sequest search engine against a human plasma protein database created from the human plasma PeptideAtlas (2012)28 using deamidation (Asn, Gln), alkylation (Cys), oxidation (Met), glycation (glucose at Lys) and advanced glycation end-products (CEL, CML) as variable modifications.


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Results MS/MS analysis of carboxymethylated and carboxyethylated model peptides Peptide 1 (H-AGSAKASGFA-NH2) and its carboxymethylated (2) and carboxyethylated (3) versions were obtained after preparative RP-HPLC in high purities (> 92%), but only moderate to low yields of 79%, 33% and 3.4%, respectively. The mass spectra always displayed the singly protonated quasimolecular ions as base peaks. The tandem mass spectrum (CID-QqQ) of the unmodified peptide (m/z 865.5) displayed four y-ions (y6-y9) and the complete b-series from b3 to b10 (Figures 2A and S-1A). This signal distribution was determined by the two amino groups present at the N-terminus and Lys-5. A few signals at yn17 and bn-18 indicated weak losses of ammonia (Lys-5) or water (Ser-3 and Ser-7). Several Lys-containing internal fragment ions (simultaneous b- and y-fragmentation) were observed in the lower mass range at m/z 200.1 (AK or KA), 287.1 (SAK or KAS), 344.2 (GSAK or KASG), and 491.3 (KASGF). The lower mass region was dominated by the signal at m/z 129.1, which could correspond to the b2-ion or an internal fragment ion, i.e. protonated αamino-ε-caprolactam, which is often obtained for lysine-containing peptides29 (Figure 1B). As this signal was not detected for CML and CEL, as described below, it derives most likely from the lysine residue and does not represent the b2-ion. The weak signal at m/z 84.1 potentially represents protonated tetrahydropyridine (C5NH10) resulting either from protonated α-amino-ε-caprolactam (-45 m/z-units, losses of NH3 and CO) or from protonated pipecolic acid at m/z 130.1 (-46 m/z-units, losses of H2O and CO). It needs to be mentioned though that this latter signal was not detected (Figures 2A and S-1A)29. The mass spectra of the corresponding CML- and CEL-containing peptides were very similar to the unmodified sequence indicating that alkylation of Lys did not have a major impact on the fragmentation pattern and that the carboxyl groups altered the intensities of the fragment ions only slightly (Figures 2B, 2C, S-1B, and S-1C). The increment masses of 186 and 200



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from b4 to b5 and y5 to y6 (only CEL-modified peptide) unambiguously identified CML and CEL, respectively, in position 5. Relative to the unmodified peptide, the b5- to b10-ion series were shifted by 58 and 72 m/z-units, which further confirmed the modifications. It is important to note that CML and CEL residues were stable under CID, a finding that was also reported for acetylated and hexanal-modified lysine residues.30,31 The above-mentioned four internal fragment ions were repeatedly detected with a mass increase of 58 and 72 m/z-units confirming CML and CEL, respectively (Figures 1B and 1C). These signals were detected at m/z 258.2, 345.2, 402.2, and 549.3 (peptide 2) as well as m/z 272.2, 359.2, and 416.2 (peptide 3). The Lys-derived fragment ions at 84.1 and 129.1 were not detected, but were replaced by two signals at m/z 142.1 and 187.1 appearing for peptide 2 and at m/z 156.1 and 201.1 for peptide 3. The mass shifts of 58 and 72 m/z-units relative to the unmodified peptide correspond to the carboxymethyl- and carboxyethyl-groups, respectively, indicating modified α-amino-εcaprolactam and tetrahydropyridine structures (Figures 1B and 1C). Interestingly, the signals corresponding to the suggested amino-ε-caprolactam structures were very intense providing a good basis for a precursor ion scan. This was also true for the enhanced product ion mode (EPI) on the QqLIT and for tandem mass spectra acquired on a QqTOF-MS using CID fragmentation (Figure S-2) or LTQ-Orbitrap-MS with HCD functionality (Figure S-3). Precursor ion scan The mass spectrometric parameters were optimized to obtain high intensities of precursor (Q1) and characteristic reporter fragment ions (Q3) using crude synthetic CML- and CELmodified peptides. The fragment ions corresponded to HSA sequences 349-359 (ERAFKCM/CEAWAV-NH2; peptides 4 and 5), 525-534 (KCM/CEQTALVELVK; peptides 6 and 7), and 208-216 (LAKCM/CETYETTLEK; peptides 8 and 9) (Table 1). The tandem mass spectra of all six peptides displayed the α-amino-ε-caprolactam-related signal at high intensities and the tetrahydropyridine-signal typically at lower intensities (Figures 3, S-4). 10 Environment ACS Paragon Plus

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For low curtain gas pressure (CUR, 10 psig) maximum intensities were achieved for all peptides at declustering potential (DP) values of 50 to 80 V (Figure S-5). As this setting resulted in high in-source fragmentation, the DP was lowered to 25 V despite reducing the signal intensities by 50% to 85% (Figure S-6). The optimal collision potential (CE) varied from 35 to 60 V depending on the peptide, precursor ion charge state, and reporter ion. The αamino-ε-caprolactam-related signals at m/z 187.1 and 201.1 were most intense for CE values of 30 to 35 V for doubly charged and 40 to 55 V for triply charged CML- and CEL-containing peptides (Figure S-7). CE values between 20 and 30 V higher CE values were optimal for the detection of tetrahydropyridine-related signals at m/z 142.1 and 156.1 (Figure S-7), which were for peptide 7 even two- to threefold more intense than the α-amino-ε-caprolactamrelated signals (Figure S-7A). The latter signals, however, might generally be more useful as their optimal CE values depended little on the sequence, which can be explained by the lower number of consecutive fragmentation reactions.29 In this case both precursor ion scans were combined to increase the reliability of the analysis. The Q1 scanning step size was 0.1 m/z to achieve the highest possible mass accuracy. When varying the dwell times from 10 ms to 2 s, we observed the highest signal intensity for peptide 2 at 0.2 s.

Method validation The optimized methods were validated in terms of sensitivity using a dilution series of peptides 2 and 3 (1 – 10 nmol/L), which were infused in the QqLIT-MS at a flow rate of 5 µL/min. The limits of detection (LODs) were 5 nmol/L and 2.5 nmol/L for peptides 2 and 3 in 60% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid, respectively (instrument detection limit, LODi; Figure S-7, Table S-3),32 and 20 and 50 nmol/L (method detection limit, LODm) when spiked into a tryptic digest of HSA (0.5 µmol/L, Figure S-8, Table S-3). As both LODi and LODm were independent of the resolution modes “high” (wh = 0.4 to



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0.6 m/z-units) and “unit” (wh = 0.6 to 0.8 m/z-units) used for Q1 and Q3, both mass analyzers were set to “high” resolution modes in all following analyses. Both tetrahydropyridine-related reporter ions (m/z 142.09 and 156.10) differed by at least 1 m/z-unit for all theoretically calculated masses of singly charged yn-, bn-, immonium, and internal b-/y-fragment ions of unmodified peptides. In contrast, the α-amino-ε-caprolactamrelated signals (m/z 187.11 and 201.12) were close to the m/z-values calculated for several b2ions (Table S-4), and could only be resolved by high-resolution mass analyzers.

CML- and CEL-modification sites in glycated HSA Precursor ion scans acquired at m/z 142.1 and 187.1 by direct infusion to detect CML- and at m/z 156.1 and 201.1 to detect CEL-modified peptides showed many intense signals in the mass range from m/z 300 to 700 for a tryptic digest of heavily glycated HSA (Figure S-6 and S-10). As both modifications and glycation can prevent tryptic cleavage at the modified residues, we expected to detect longer peptides with higher charge states.33 The 50 most intense signals were used to create an inclusion list for a targeted DDA (± 0.4 m/z-units) for the ensuing nano-UPLC-ESI-LIT-Orbitrap-MS analysis (Figure 5). The data sets were processed with Sequest against a human plasma protein database assuming that the precursor ions were either doubly or triply protonated, as the charge state could not be retrieved from the precursor ion scan. Hits with Xcorr-values lower than 2.2 for doubly and 3.75 for triply charged precursor ions were confirmed manually. All unmatched signals were recalculated assuming charge states of four or five (instead of two or three), included in a second inclusion list (± 0.4 m/z-units), and re-analyzed by targeted DDA. Eight peptides representing seven CML sites but no CEL modifications were assigned in the first round and only one additional CML site in the second analysis (Table 2, Figure 5). Peptide KCMQTALVELVK (CML at position 525; m/z 396.237) displayed a relatively intense signal (Figure 4B) indicating a high modification degree, which was also shown for the corresponding Amadori-product.34 Seven 12 Environment ACS Paragon Plus

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CML positions were identified by both reporter ions. Although the scan at m/z 142.1 was less sensitive, it was more specific for CML and yielded only one incorrect assignment compared to three for m/z 187.1 (Table S-5).

CML-modified plasma proteins Plasma samples obtained from three type 2 diabetes mellitus (T2DM) patients were pooled, digested and analyzed by direct infusion for CML-containing peptides as the most abundant lysine-derived plasma AGE.1 Both reporter ions yielded many signals, with m/z 187.1 providing again more intense signals (Figure 6), of which the 50 most intense signals were analyzed as described above (Figure 5). Eighteen CML-containing peptides corresponding to 13 m/z-values of the precursor ion scan (some peptides were isobaric) were assigned with high confidence (Xcorr ≥ 2.20 for z = 2 and ≥ 3.75 for z = 3, Table 3) representing 21 CMLsites in 17 proteins. Among the remaining peptide sequences retrieved with low confidence scores, 21 peptides were manually confirmed. Additionally, 10 signals of the precursor ion scan did not contain a CML-residue, but displayed fragment ions at m/z 187.1 that correspond to calculated masses of b- or internal ions (Table S-6). Surprisingly, only two CML-sites were identified in HSA (K64 and K371), whereas all other modifications were detected in 16 proteins of low to medium abundance, such as cytoskeleton and associated (6), nuclear (4), and signal transduction (2) proteins (Table 3).



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Discussion Fragmentation characteristics of CEL- and CML-containing peptides Mass increments of 58 and 72 Da at Lys- and Arg-residues indicating carboxymethylation and carboxyethylation, respectively, can be used as variable modifications in database search.35 However, specific reporter ions or indicative fragmentation patterns, such as neutral losses or internal fragment ions, improving the confidence level in DDA-based MS/MS36 have not been reported yet. We started with the short model peptide H-AGSAKASGFA-NH2 containing only one Lys residue in position 5. In this model sequence, amino acid residues prone to backbone fragmentations, such as Pro, His and C-terminal Lys or Arg,37,38 were omitted. Importantly, only minor signals indicating water or ammonia losses were present in the tandem mass spectrum allowing an easy spectral interpretation. The internal fragment ions produced by CEL- and CML-containing peptides resulted most likely from the y6-ion by an intra-molecular nucleophilic attack of the alkylated ε-amino group on the carbonyl group of the peptide bond similar to the diketopiperazine peptide fragmentation pathway.39 Similarly, y7- or y8-ions could yield internal fragment ions containing a C-terminal Lys residue. This was further confirmed by peptides 4 to 9 of HSA (Figures 3, S-4). The detected Lys-, CMLand CEL-related immonium ions are likely formed by similar mechanisms29 (Figure 1). Interestingly, signals at m/z 142.1, 156.1, 187.1, and 201.1 were intense in all acquired tandem mass spectra independent of the instrument type (i.e. QqQ-, QqLIT-, QqTOF- and LTQ-Orbitrap-MS), though it must be noted that all experiments relied on CID in a quadrupole. Therefore, all four α-amino-ε-caprolactam- and tetrahydropyridine-related signals seem to be suitable for a precursor ion scan, as applied for other ε-substituted lysyl residues including Amadori compounds. 30,40,31

Precursor ion scan


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Due to the lack of proteomics studies reporting CML- and CEL-modification sites in plasma proteins, we had to consider dominant glycation sites reported for abundant plasma proteins,34,41 as Amadori products were reported to be direct precursors of CML.42 Thus, we assumed that this autoxidation product and CEL should be present at heavily glycated residues in vivo. Consequently, peptides containing the dominant glycation sites of HSA at K525 and K351 were synthesized to optimize the parameters of a precursor ion scan from m/z 300 to 900, a process that was completed within 20 min. The resulting m/z-values indicating CML- or CEL-modified peptides will allow a fast identification by LC-MS in targeted DDAmode. This strategy is significantly faster than identifying modified peptides in complex sample mixtures, which typically comprise multiple LC-MS analyses with shallow gradients to identify modified peptides by gas phase fractionation (GPF) or two-dimensional separations. We have also considered a possibility of designing a single LTQ-Orbitrap-MS DDA method with a precursor ion scan for specific m/z as survey sub-experiment and a product ion LIT scan as a dependent one. However, we refused of using this setup, because of compromised sensitivity in the survey scan and, hence, expected lower modification coverage. The strong influence of the tryptic serum matrix on the LODm, which was 4-20-fold lower than the LODi is most likely related to ion suppression effects, which is common for enzymatic digests of plasma or tissue samles.43 This is especially true for the low signal intensities obtained for AGE-modified peptides present only at very low quantities. The LODm might be improved by adding an additional chromatographic separation orthogonal to RPC, though recovery rates and splitting of a peptide in several fractions (if coupled off-line) will also reduce the sensitivity. Alternatively, increasingly sensitive mass spectrometers can be used that should provide 10 to 50 times lower LODms (and LODis).

CML- and CEL-sites in glycated HSA and plasma 15 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The mass signature obtained for the CML-specific reporter ions was mostly independent from the peptide (Figure 4), with the signal at m/z 187.1 typically being most intense, relating to its favorable formation and a cross-talk to b2- and internal fragment ions (Table S-4). Expectedly, it was impossible to identify the corresponding peptides by targeted MS/MS infusion experiments (data not shown) due to the high sample complexity and the low signal intensities obtained for AGE-candidates. Therefore, we complemented precursor ion scanning with targeted LC-MS/MS approach. In order to simplify data interpretation, in this “prove of the concept” study we selected only the 50 most intense signals. However, this number can be extended for analysis of biological samples. The LC-MS yielded eight CML-sites, more than previously reported for glycated HSA.44,45,46,47 Only the K106 and K525 CML-sites were reported before, with K525 being expected to be the dominant site as it is also the major glycation site.34 The remaining six CML-sites have not been reported before, though they are all known to be glycated by glucose in vitro and in vivo.34,41 Other sites reported for minimally glycated HSA, such as K205, K564 and K286,48,47 were not detected. Probably these residues are involved in additional oxidation and cross-linking reactions. This assumption is supported by several intense signals present in both CML-specific precursor ion scans, which could not be assigned to the HSA sequence based on their mass and tandem mass spectra (Figure 4). However, it was beyond the scope of this study to identify other AGEs. Surprisingly, no CEL-modified HSA peptides were identified by nano-LC-Orbitrap-MS/MS. This might indicate significantly lower contents, which could be related to the underlying reactions, i.e. direct alkylation of Lys rather than degradation of an Amadori product.6 Indeed, the targeted m/z-values were not detected in the nano-UPLC-Orbitrap-MS. In diabetic plasma samples, only two CML-sites (K64 and K371) reported earlier were identified in HSA,41 which can be explained by the much lower glucose concentrations in diabetic patients (10 µmol/L) than used for in vitro glycation (0.5 mol/L). Surprisingly, no 16 Environment ACS Paragon Plus

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further abundant proteins were found to be carboxymethylated. However, some tissue proteins with relatively long (in comparison to major plasma polypeptides) half-life times and high lysine contents, such as contractile molecules, were easily detected in plasma49,50 and thus appear also to contain more CML (Table 3). Signal transduction proteins like receptors, protein kinases and transcription factors are well accessible to proteomics studies,50 with high CML-levels (Table 3). Considering their origin (probably platelet) close to membrane compartments, might indicate direct carboxymethylation by glyoxal derived from lipid peroxidation.51 Among the 17 CML-modified proteins, only seven were recently reported as glycated in plasma by Zhang et al. indicating that many CML-sites can not be deduced from known glycation sites but are either formed by other reaction pathways or are oxidatively degraded very fast.

Conclusions CID yielded for both CML- and CEL-modified peptides two unique and modification-specific signals (i.e. tetrahydropyridine and α-amino-ε-caprolactam) at m/z 142.1 and 187.1 (CML) and m/z 156.1 and 201.1 (CEL). The precursor ion scans established for these sequenceindependent reporter ions provided LODi of 2.5 nmol/L for CML- and 5 nmol/L for CELcontaining peptides (sample infusion). When combined with targeted nano-UPLC-OrbitrapMS, eight CML-modifications were identified in glycated HSA at lysine residues 12, 106, 190, 199, 351, 414, 475, 525. In pooled plasma sample obtained from T2DM patients, 21 CML-modifications sites were identified in 17 proteins including K88 and K396 in HSA. Thus, the presented strategy is capable of screening plasma samples for CML-modified biomarkers, especially if more sensitive mass spectrometers are used.

Acknowledgements



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

We thank Prof. Matthias Blüher and Rico Schmidt for providing plasma samples and a tryptic digest

of

glycated

HSA,

respectively.

Financial

support

from

“Deutsche

Forschungsgemeinschaft” (DFG, HO-2222/7-1), European Fund for Regional Structure Development (EFRE, European Union and Free State Saxony), “Bundesministerium für Bildung and Forschung” (BMBF), and Free State Saxony to RH as well as a scholarship of Ernst-Schering Foundation to UG are gratefully acknowledged.


Page 19 of 38

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Tables Table 1: Numbering and sequences of synthetic peptides. Peptide

Sequence

Origin

1

H-AGSAKASGFA-NH2

2

H-AGSAKCMASGFA-NH2

3

H-AGSAKCEASGFA-NH2

4

H-ERAFKCMAWAV-NH2

HSA(208-216)

5

H-ERAFKCEAWAV-NH2

not reported to be glycated in vitro or in vivo

6

H-KCMQTALVELVK-OH

tryptic peptide, HSA(525-534)

7

H-KCEQTALVELVK-OH

glycated in vivo34 and carboxymethylated in vitro52

8

H-LAKCMTYETTLEK-OH

9

H-LAKCETYETTLEK-OH

Artificial sequence

tryptic peptide, HSA(349-359) glycated in vivo34



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

Table 2: AGE-modified peptides identified by a precursor ion scan in a tryptic digest of glycated HSA and their annotation by RPC-Orbitrap-MS. Precursor ion scan No.a

m/z

Reporter ion rel. int. [%]

nanoUPLC-LIT-Orbitrap-MS (DDA) tR [min]

m/z

z

Xcorrb

Prob.

Sequencec

Modified residue

187.1

142.1

1

336.0

5

-

17.4

335.850

3

2.33

1.00

LKCMC*ASLQK

K199

2

395.3

12

18

18.3

394.948

3

1.22

1.00

LDELRDEGKCMASSAK

K190

3

396.4

5

37

28.7

396.237

3

4.69

7.63

KCMQTALVELVK

K525

5

429.0

7

19

26.7

428.872

3

3.52

1.72

FKCMDLGEENFK

K12

6

452.3

6

12

23.2

452.238

3

3.71

1.00

LAKCMTYETTLEK

K351

7

509.3

6

6

21.4

508.906

3

4.04

1.00

VTKCMC*C*TESLVNR

K475

8

539.4

6

-

18.9

539.593

3

2.38

1.00

LDELRGlDEGKCMASSAK

K190

9

566.8

5

14

29.9

566.649

3

4.01

49.16

KCMVPQVSTPTLVEVSR

K414

10

642.9

5

-

26.9

642.805

2

3.05

21.63

FKCMDLGEENFK

K12

11

429.8

7

-

21.4

572.598

3

1.29

1.00

QEPERNECFLQHKCM

K106

1.60

1.00

QEPERCMNECFLQHK

R98

a

Peptides are listed in order of increasing m/z-values.

b

Sequences with Xcorr-values below 2.20 and 3.75 for doubly- and triply-charged precursor ions, respectively, were confirmed manually.

c

KCE, KCM, C*, RGl , and RCM denote CEL, CML, carbamidomethylated Cys, Glarg-modified Arg, and CMA, respectively.


Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Table 3 AGE-modified peptides identified in the tryptic digest of pooled human T2DM plasma using first a precursor ion scan and then targeted nanoLC-LIT-Orbitrap-MS/MS. Precursor ion scan Reporter ion Noa m/z

rel. int. [%] 187.1

142.1

nanoLC-LIT-Orbitrap-MS DDA

Detected at m/z

Protein annotation tR

b

PIS

c

lower z

(min)

m/z

z

XCorrd

Prob.

Annotated sequencee

Accession number

Protein description Disheveled-associated activator

1

339.9

9

3 → 2+

-

30.4 509.296 2

2.26

1.0

KCMSLLALEKCM

Q9Y4D1

of morphogenesis 1 [DAAM1_HUMAN]

Sitef

K117, K124

Transcription initiation factor 2

387.3

5

3 → 2+

-

25.9 580.279 2

2.33

1.0

AKCMMRMG-HEQER

Q8IZX4

TFIID 210 kDa subunit

K667

[TAF1L_HUMAN] 3

4

440.8

480.8

18

-

4

X

27.2 440.724 2

2.22

1.0

X

26.2 480.759 2

2.57

1.0

QDEAMAFAKCMPDIOXK KCMQDEAMQLTGAR

Q6IBS0

Q9Y6X6

49

Twinfilin [TWF2_HUMAN] Myosin-XVI [MYO16_HUMAN]

K325

K565

Histone-lysine NX

26.2 480.759 2

2.20

1.0

KCMPOXSPOXSKAR

Q8TEK3

methyltransferase, H3 lysine-79

K388

specific [DOT1L_HUMAN] 5

500.7

11

-

3 → 2+

15.8 750.285 2

3.66

16.2

TCCAMVADESAENCDKCM


P02768

Serum albumin precursor [ALBU_HUMAN]

K88


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

6

508.4

8

-

X

34.4 508.311 2

2.34

1.0

KCMLGKVAPOXTK

Page 22 of 38

Q9H720

PGAP2-interacting protein [PG2IP_HUMAN] Disheveled-associated activator

X

31.7 509.296 2

2.42

1.0

KCMSLLALEKCM

Q9Y4D1

of morphogenesis 1 [DAAM1_HUMAN]

7

509.4

10

5

X

31.7 509.296 2

2.39

1.0

X

31.6 509.296 2

2.33

1.0

X

31.7 509.296 2

2.30

1.0

KOXELLALEKCM IKDIOXIIAPOXPDIOXKCM ROXRCMIANKKCM

K418

K117, K124

Q92817

Envoplakin [EVPL_HUMAN]

K1065

Q9BYX7

Kappa-actin [ACTK_HUMAN]

K334

O43739

Cytohesin-3 [CYH3_HUMAN]

K400

IQ and AAA domainX

35.1 509.800 2

2.29

1.0

KDLTKCMALROX

A6NCM1

containing protein ENSP00000340148

K655

[IQCAL_HUMAN]

8

9

517.4

518.4

20

4

-

-

X

25.4 517.766 2

3 → 2+

3 → 2+ 10 547.4

15

18.8 777.301 2

34.6 820.872 2

2.41

1.0

2.42

1.0

2.54

1.0

4 3 → 2+

34.6 820.872 2

2.34

1.0

AMOXAIADALGKCM CCAMCCAMAAADPHECYAKCM YFEKCMKCMWOXTDTFOXK KCMYFSTCCAMKCMNWYK

22 ACS Paragon Plus Environment

UDP-glucuronosyltransferase Q9HAW8

1-10 precursor

K325

[UD110_HUMAN] P02768

Serum albumin precursor [ALBU_HUMAN] Bromodomain adjacent to zinc

Q9UIF8

finger domain protein 2B [BAZ2B_HUMAN]

Q15063

K396

K2159, K2160

Periostin precursor

K55,

[POSTN_HUMAN]

K61

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


11 552.9

4

-

X

28.1 552.799 2

2.25

1.0

KCMEARDEAMQDEAMLKOXR

Peptidyl-prolyl cis-trans Q6UX04

isomerase SDCCAG10

K318

[SDC10_HUMAN] Transcription initiation factor

12 580.4

4

-

X

27.1 580.278 2

2.26

1.0

AKCMMRMG-HEQER

Q8IZX4

TFIID 210 kDa subunit

K667

[TAF1L_HUMAN]

X

13 644.7

4

-

25.5 644.308 2

2.25

1.0

DYKCETMOXTALAKCM

cAMP-dependent protein P31321

22.2 644.338 2

2.24

1.0

KCMEKLWESPGR

Q96JG9

X

22.2 644.338 2

2.27

1.0

KEKCMLWESPGR

Q96JG9

Peptides are numbered by increasing m/z values.

b

Originally detected in the modification-specific precursor ion scan.

c

m/z recalculated to lower charge state.

d

Only peptides annotated with Xcorr ≥ 2.20 for doubly- and ≥ 3.75 for triply charged ions are listed.

e

CM,

f

Numbering for the longest listed protein sequence including N-terminal Met and signaling peptides.

Zinc finger protein 469 [ZN469_HUMAN] Zinc finger protein 469 [ZN469_HUMAN]

carboxymethylation; CE, carboxymethylation; OX, oxidation; DIOX, double oxidation; CAM, carbamidomethylation; DEAM, deamidation.

23 ACS Paragon Plus Environment

K128

subunit [KAP1_HUMAN]

X

a

kinase type I-beta regulatory

K2230

K2232


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure legends Figure 1: Structures of lysine (R1), Nε-(carboxymethyl)lysine (CML, R2), and Nε(carboxyethyl)lysine (CEL, R3) (A) and their respective α-amino-ε-caprolactam (B) and tetrahydropyridine reporter ions (C).

Figure 2: Tandem mass spectra of m/z 865.5 (A), 923.5 (B), and 937.5 (C) corresponding to [M+H]+ ions of peptides 1, 2, and 3, respectively, recorded on the QTrap in QqQ mode (collision potential 50 V). Signals indicating neutral losses of water and ammonia are marked with asterisks (*) and hashtags (#), respectively, and α-amino-ε-caprolactam- and tetrahydropyridine-related lysine-derived ions are labeled with full diamonds (♦) and circles (●), respectively. Signals indicating internal fragment ions are denoted as inta-b (a and b as terminal residues).

Figure 3: Tandem mass spectra of m/z 677.8 and 684.9 corresponding to doubly protonated peptides 8 (A), and 9 (B). Mass spectra were recorded on a QqLIT-MS in enhanced mode applying a collision potential of 35 V. Signals indicating internal fragment ions are denoted as inta-b (a and b as terminal residues). Full diamonds (♦) and circles (●) denote signals of αamino-ε-caprolactam and tetrahydropyridine reporter ions, respectively.

Figure 4: Precursor ion scans acquired for m/z 187.1 (A) and 142.1 (B) in a tryptic digest of glycated HSA, which was directly infused (5 µL/min) into the mass spectrometer. Signals of confirmed CML-containing peptides are marked with an asterisk (*, Table 2), signals from non-CML-peptides are marked with hashtags (#, Table S-5).

Figure 5: Flow chart of the strategy applied for the detection and identification of CML- and CEL-modification sites in proteins. ACS Paragon Plus Environment

Page 24 of 38

Page 25 of 38

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Figure 6: Precursor ion scans acquired by direct infusion (5 µL/min) on the QqLIT-MS for m/z 187.1 (A) and 142.1 (B) in a tryptic digest of diabetic human plasma. Signals for which CML-containing peptides were identified are marked with an asterisk (*, Table S-6), signals from non-CML-peptides are marked with hashtags (#, Table S-7).



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(10) Portero-Otin, M.; Pamplona, R.; Bellmunt, M.J.; Ruiz, M.C.; Prat, J.; Salvayre, R.; Negre-Salvayre, A. Advanced glycation end product precursors impair epidermal growth factor receptor signaling. Diabetes 2002, 51, 1535-1542. (11) Nilsen, B.M.; Berg, K.; Arukwe, A.; Goksoyr, A. Monoclonal and polyclonal antibodies against fish vitellogenin for use in pollution monitoring. Mar Environ Res 1998, 46, 153-157. (12) Ahmed, N.; Argirov, O.K.; Minhas, H.S.; Cordeiro, C.A.; Thornalley, P.J. Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with

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Figure 1 115x185mm (300 x 300 DPI)


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Figure 2 382x958mm (300 x 300 DPI)



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Figure 3 160x68mm (300 x 300 DPI)


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Figure 4 258x364mm (300 x 300 DPI)



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Figure 5 174x266mm (300 x 300 DPI)


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Figure 6 253x349mm (300 x 300 DPI)



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TOC 49x29mm (600 x 600 DPI)


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Sensitive and Site-Specific Identification of Carboxymethylated and

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