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Profiling of Thiol-Containing Compounds by Stable Isotope Labeling Double Precursor Ion Scan Mass Spectrometry Ping Liu, Yun-Qing Huang, Wen-Jing Cai, Bi-Feng Yuan,* and Yu-Qi Feng* Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China S Supporting Information *

ABSTRACT: Here we developed a novel strategy of isotope labeling in combination with high-performance liquid chromatography−double precursor ion scan mass spectrometry (IL−LC−DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of isotope labeling reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-d7 labeled thiol-containing compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiolcontaining compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, offering good identification and accurate quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Using the IL−LC−DPIS-MS strategy, we profiled the thiolcontaining compounds in beer and human urine, and 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with thiol standards or tandem mass spectrometry analysis. Taken together, the IL−LC−DPIS-MS method is demonstrated to be a promising strategy in the profiling of compounds with identical groups in metabolomics study.

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polar and ionic metabolites that normally are poorly retained on reversed-phase column, and the ionization efficiencies of metabolites could also be improved upon isotope labeling due to the addition of ionizable functional groups. Moreover, the isotope-labeled analogues were often used as the internal standards to overcome the matrix and ion suppression effect.11 The typical method of differential isotope labeling normally introduces a light isotope tag to the analytes in one sample and a heavy isotope tag to another comparative sample, followed by mixing the light and heavy isotope-labeled samples for MS analysis.11 The metabolites with common a functional group can be easily recognized through determination of a chromatographically coeluted pair of isotope-labeled analytes (MS doublet peaks) with characteristic mass difference.13 The isotope-labeled analytes pair can provide the information on relative quantification as well as the identification of the metabolites. For example, Guo et al. used 13C- and 12C-dansyl

ith the rapid advancement of analytical methods, metabolomics has been widely applied in various research fields, including disease diagnosis,1,2 food science,3,4 and drug discovery.5,6 Mass spectrometry (MS) is one of the most prominent platforms for metabolomics study due to its high sensitivity and specificity.7 However, profiling of unknown compounds remains to be a bottleneck of MS-based metabolomics.8 First, certain compounds are inherently difficult to analyze by liquid chromatography−electrospray ionization mass spectrometry (LC−ESI-MS) and may thus escape detection. For example, some compounds are too hydrophilic to be sufficiently retained on common reversed-phase LC columns, or have poor ionization yield in ESI due to the absence of ionizable functional groups, which leads to the inefficient detection by MS.9 Second, the quantitative profiling of metabolites by MS is still challenging because the MS responses of metabolites fluctuate and ionization efficiencies alter even under the same liquid chromatography conditions.10 To circumvent these problems, the chemical isotope labeling (IL) strategy has been developed for qualitative or quantitative profiling of metabolites with the MS-based platform.11,12 Isotope labeling can alter the chromatographic behaviors of © 2014 American Chemical Society

Received: June 26, 2014 Accepted: September 15, 2014 Published: September 15, 2014 9765

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of the BQB and BQB-d7 labeled thiol-containing compounds, and the peak pairs with characteristic mass differences can be extracted from the two PIS spectra and assigned as potential thiol-containing candidates. The detected thiol-containing candidates were further identified by product ion scan and high-resolution mass spectrometry analysis.

chloride to derivatize amine, and phenolic hydroxyl group where the differential isotope tags can be introduced for qualitative and quantitative profiling of compounds in biological samples.13,14 Yang et al. described an LC−MS method for γtocopherol analysis in human A549 cell by chemical derivatization with N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NANHS) or (C1-NANHS- d3).15 The strategy of IL combined with MS facilitates the identification of metabolites. However, there are several disadvantages of this method performed under full scan mode. First, the isotopic cluster peaks of unlabeled abundant metabolites may overlap with the peaks of labeled metabolites, which increases the difficulty of peak interpretation. In addition, the dissociation of molecular ions of metabolites in the interface followed by translation to the mass analyzer will cause ambiguous assignment of mass spectra peaks under full scan mode.16 Moreover, a number of peaks may arise from the fragment ions of metabolites rather than the intact ions of metabolites, and the numbers of ions detected in multiple experiments may not be identical.16 Compared with full scan mode, precursor ion scan (PIS) that monitors the characteristic fragment ions produced by the fragmentation of precursor ions can provide better detection sensitivity with lower noise due to the improved selectivity.17 PIS mode is powerful in the analysis of molecules which possess one identical moiety and similar fragmentation pattern.17 However, the requirement of the identical moiety of analytes limits the application of PIS in metabolomics study. In this respect, chemical labeling may expand the application of PIS. For example, O’Brien-Coker et al. used cyclohexanedione to derivatize aldehydes, and the common fragment ion at m/z 216 was employed using PIS to detect all cyclohexanedionederivatized species.18 Boughton et al. used 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate to derivatize monoamines, and the common fragment ion of derivatives at m/z 171 was used by PIS to detect all monoamine-containing metabolites.19 Therefore, chemical labeling in combination with PIS could be a promising strategy to qualitatively and quantitatively profile a relatively wide range of metabolites that are normally inefficiently detected by LC−ESI-MS. Thiol-containing compounds, which belong to the group of nonideal LC−ESI-MS analytes, are important for the various aroma impacts of beer and wine.20,21 In addition, thiolcontaining compounds can potentially inhibit nonenzymatic oxidative reactions, which cause the development of off-flavors during beer aging.22 Therefore, profiling of thiol-containing compounds is important to evaluate organoleptic characteristics and nutrition of beer. Here we developed a novel strategy for nontargeted profiling of thiol-containing compounds in beer by isotope labeling combined with high-performance liquid chromatography− double precursor ion scan mass spectrometry (IL−LC−DPISMS) analysis. In this strategy, we synthesized a pair of isotope labeling reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group, and they have been demonstrated to selectively react with thiol-containing compounds.24 The major advantage of this strategy is that two characteristic product ions (m/z 218 and 225 containing isotope tag) assigned to the fragmentation at the C−S bond were generated from the BQB and BQB-d7 labeled thiol-containing compounds, respectively. In this regard, DPIS (m/z 218 and 225) was used for the profiling

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MATERIALS AND METHODS Chemicals and Reagents. N-Acetyl-cysteine (Nac), glutathione (GSH), 3-mercaptohexan-1-ol (3-MH), glycine, quinoline, quinoline-d7 were purchased from Sigma (St. Louis, MO, U.S.A.). Chromatographic grade methanol and acetonitrile (ACN) were purchased from TEDIA Co. Inc. (Ohio, U.S.A.). Formic acid and ethylenediaminetetraacetic acid (EDTA) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other solvents and chemicals used were of analytical grade. The water used throughout the study was purified by a Milli-Q apparatus (Millipore, Bedford, MA). Tsingtao Beer (Qingdao, China) was obtained from a local store in Wuhan, China. Stock standard solutions of Nac, GSH, and 3-MH were prepared in 1.0 mmol/L ethylenediaminetetraacetic acid (EDTA) solution containing 0.05% formic acid at a concentration of 1.0 mmol/L. Synthesis of BQB and BQB-d7. ω-Bromoacetonylquinolinium bromide (BQB) and BQB-d7 were synthesized according to our previous work.23 Briefly, bromine was added dropwise to a round-bottom flask containing acetone and methanol to form 1,3-dibromoacetone. Then, quinoline or quinoline-d7 dissolved in toluene was added dropwise to the solution of 1,3-dibromoacetone for 2 h at 0 °C with stirring. The reaction mixture was then stirred overnight at room temperature. The precipitate was collected and recrystallized with absolute ethanol and dry ethyl ether. Stock solutions of BQB and BQB-d7 were prepared in acetonitrile at the concentration of 10 mmol/L, respectively, and stored at −20 °C. BQB Labeling Reaction. The reaction for isotopic labeling of thiol-containing compounds using BQB and BQB-d7 is shown in Figure 1. Briefly, 30 μL of BQB or BQB-d7 (1 mmol/

Figure 1. Derivatization reaction for thiol-containing compounds using BQB and BQB-d7.

L) was added into a 1.5 mL tube and dried under nitrogen gas. Subsequently 200 μL of Gly−HCl buffer solution (5.0 mmol/L, pH 3.5) and 100 μL of beer were added. The mixture was incubated at 60 °C for 60 min with shaking at 1500 rpm. Then equal volumes of BQB and BQB-d7 labeled sample solutions were mixed and 50 μL of the solution was used for analysis by LC−DPIS-MS. LC−DPIS-MS Analysis. Analysis of samples was performed on the LC−ESI-MS/MS system consisting of an AB 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, 9766

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scans were detected in the mass range of m/z 50−550 using dynamic fill and a scan rate of 4000 Da/s. LC−QTOF-MS Analysis. High-resolution mass spectrometry experiments were performed on the LC−QTOF-MS system consisting of a MicrOTOF-Q orthogonal-accelerated time-offlight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) with an ESI source (Turbo Ionspray) and a Shimadzu LC-20AB binary pump HPLC (Tokyo, Japan), a SIL-20AC auto sampler, and a DGU-20A3 degasser. Data acquisition and processing were performed using Bruker Daltonics Control 3.4 and Bruker Daltonics Data analysis 4.0 software. The HPLC separation column and mobile phase gradient were same as that of the LC−DPIS-MS method. The mixture of BQB and BQB-d7 labeled samples (1/1, v/v) were detected under positive ion mode. The optimized ESI parameters were as follows: capillary voltage, −4.5 kV; dry gas, 5.0 L/min; dry temperature, 180 °C; funnel 1 rf, 200.0 Vpp; funnel 2 rf, 200.0 Vpp; ISCID energy, 0.0 eV; hexapole rf, 200.0 Vpp; prepulse storage, 12.0 μs. Spectra were acquired by summarizing 5000 single spectra. Full scan mode was used. Extraction of centroid spectra peaks with a width of 0.01 Da was used to pick up the extracted ion chromatograms (EICs) from the total ion chromatogram (TIC). The prospective molecular formulas of BQB-thiol derivatives were generated based on the accurate mass and isotope patterns of elemental composition using Bruker Daltonics Data analysis 4.0 software. A mass tolerance of 5.0 mDa was set, and a maximum elemental composition of C = 50, H = 100, N = 50, O = 50, S = 10, P = 10, and Cl = 10 was used. The molecular formulas of thiol-containing compounds were obtained by subtracting the molecular formula of BQB (C12H10NO). The molecular formulas obtained by TOF was further searched in the database of METLIN (http://metlin.scripps.edu/index. php) for putative identification. Effect of H 2 O 2 Treatment on Thiol-Containing Compounds in Beer. Thiol-type compounds, an important class of antioxidants, could be easily oxidized by oxidizing reagents. Here we used H2O2 to treat the beer to evaluate the quantitative analysis of the DPIS method by examining the contents change of thiol-containing compounds. Beer sample was divided in two aliquots, then to one was added H2O2 (1%, v/v, treated), and to the other one was added an equal volume of H2O as a control. The two samples were labeled with BQB and BQB-d7 to form the forward and reverse labeling sample solutions. In the forward labeling, the untreated beer sample was labeled with BQB and the treated sample was labeled by BQB-d7. In the reversed labeling, the untreated beer sample was labeled with BQB-d7 and the treated sample was labeled by BQB. Then the two labeled samples were mixed (1:1, v/v) and analyzed by LC−DPIS-MS. Triplicate measurements were performed in each labeling strategy. Profiling of Thiol-Containing Compounds by LC− DPIS-MS in Human Urine. The first morning urine samples were collected from 10 healthy volunteers. The urine samples were pretreated according to a previously described method.23 Briefly, 200 μL of each urine sample was added to a prepared screw-cap vial (1.0 mL) containing 18 μL of EDTA (10 mmol/ L) and 2 μL of formic acid. A pooled sample is prepared by taking an equal volume of each urine sample. Then 100 μL pooled sample was treated with 10 μL of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 10 mmol/L) under 45 °C for 60 min.

CA, U.S.A.) with an electrospray ionization source (Turbo Ionspray) and a Shimadzu LC-20AD HPLC (Tokyo, Japan) with two LC-20AD pumps, a SIL-20A auto sampler, a CTO20AC thermostated column compartment, and a DGU-20A3 degasser. Data acquisition and processing were performed using AB SCIEX Analyst 1.5 Software (Applied Biosystems, Foster City, CA, U.S.A.). The High-performance liquid chromatography (HPLC) separation was performed on a Shimadzu VPODS column (150 mm × 2.0 mm i.d., 5 μm, Tokyo, Japan) with a flow rate of 0.2 mL/min at 30 °C. Formic acid in water (0.1%, v/v, solvent A) and methanol (solvent B) were employed as mobile phases. A gradient of 0−5 min 5% B, 5− 35 min 5% to 60% B, 35−40 min 60% to 5% B, and 40−55 min 5% B was used. The DPIS method consists of two PIS (m/z 218 and 225) in the mass range of m/z 200−600 (Figure 2). A full scan was

Figure 2. Schematic diagram of the principle of IL−LC−DPIS-MS.

performed in the same mass range for comparison. DPIS and full scan were carried out under positive ion mode. The source and gas settings for both DPIS and full scans were identical. IsoSpray voltage was set at 5.2 kV, and vaporizer temperature was set at 550 °C. The mass spectrometer was operated with gas settings of 40 psi for nebulizer gas, 30 psi for curtain gas, and 60 psi for collision gas. Scan time per cycle was 2.0 s with a pause of 5.0 ms for each scan. Resolution of Q1 and Q3 was set to “low” and “unit”, respectively. Declustering potential, entrance potential, cell entrance potential, collision energy, and cell exit potential were set at 45, 7, 15, 38, and 3 V, respectively. For structural identification, IDA (information-dependent acquisition) mode was performed under positive ion mode. The IDA cycle comprised a survey scan of a PIS (m/z 218 or 225) followed by three consecutive EPI (enhanced product ion, MS/MS) scans with different collision energy offsets of 25, 35, and 45 V. The same precursor was fragmented in three EPI scans, and then the IDA cycle ended. Upon completing the cycle, a new IDA cycle started, in which the next precursor candidate was taken from an inclusion list. An inclusion list was compiled for all BQB-labeled or BQB-d7-labeled precursor ions, and the m/z and retention times were generated from the aforementioned LC−DPIS-MS analysis. The criteria were set as that EPI was triggered when signals of the preselected compounds by PIS exceeding 1000 counts/s at their retention times. The mass tolerance was set to 250 mDa, and retention time tolerance was set to 60 s. Fragments formed in the EPI 9767

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For the derivatization reaction, 50 μL of BQB or BQB-d7 (1 mmol/L) was added into a 1.5 mL tube and dried under nitrogen gas. Subsequently, 100 μL of Gly−HCl buffer solution (5.0 mmol/L, pH 3.5) and 10 μL of urine were added. The mixture was incubated at 60 °C for 60 min with shaking at 1500 rpm. Then equal volumes of BQB and BQB-d7 labeled sample solutions were mixed and 50 μL of the solution was used for LC−DPIS-MS analysis.

FMEA, which limits their applications. Due to the easy preparation of BQB and BQB-d7 as well as their favorable generation of the common characteristic fragment ion, we used BQB and BQB-d7 for the IL−LC−DPIS-MS strategy. Enhancement of Detection Sensitivity upon Chemical Labeling. The thiol-containing compounds naturally occurring in human, animals, plants, and foods, can be ionized in ESI-MS with either negative or positive ion mode. However, the ionization efficiency is normally poor. In this respect, chemical labeling of the thiol-containing compounds by BQB that harbors a precharged moiety of quaternary ammonium could largely enhance the ionization efficiencies of thiol-containing compounds during MS analysis. Here we used three thiol-containing standards (Nac, GSH, 3MH) to evaluate the enhancement of detection sensitivity upon chemical labeling by BQB. The mixture of Nac (100 μmol/L), GSH (100 μmol/L), and 3-MH (100 μmol/L) was analyzed by direct infusion under either positive or negative ion mode, and the BQB-labeled mixture (1.0 μmol/L for each analyte) was analyzed in the positive ion mode. The results showed that the signal intensities of BQB-labeled GSH increased by 500- and 80-fold compared to the unlabeled GSH detected under negative and positive ion mode, respectively (Supporting Information Figure S1). As for unlabeled Nac and 3-MH, they were barely detected by ESI-MS in either positive ion or negative ion mode due to their low ionization efficiencies, whereas after BQB labeling, they can be clearly observed even the concentration decreased 100-fold (Supporting Information Figure S1). And it was worth noting that these thiol-containing compounds were relatively stable under the reaction conditions (data not shown). Fragmentation Behavior of BQB-Labeled Thiol-Containing Compounds. Three thiol-containing standards (Nac, GSH, 3-MH) were used to investigate the fragmentation behavior of BQB-labeled thiol-containing compounds. The fragmentation behaviors of BQB-labeled thiols are shown in Supporting Information Figure S2. The results showed that two common product ions at m/z of 130.1 and 218.1 were observed for all the BQB-labeled thiols (Supporting Information Figure S2A−C). The product ion of m/z 130.1 was assigned to the fragment ion generated from the quinoline moiety, and m/z 218.1 was assigned to the fragmentation of the C−S bond in derivatives. Similarly, BQB-d7-labeled thiol-containing compounds generate the other two common product ions at m/z 137.1 and 225.1 with the same fragmentation behavior as that of BQB-labeled thiols (Supporting Information Figure S2D− F). In theory, either the ion pair of 130/137 or 218/225 can be used in the qualitative and quantitative profiling of thiolcontaining compounds in the mixture of BQB and BQB-d7 labeled samples by DPIS strategy. Selection of the Ion Pair for DPIS. As aforementioned, either ion pair of 130/137 or 218/225 can be used in the LC− DPIS-MS method for qualitative and quantitative profiling of thiol-containing compounds. Here we used the mixture solution of Nac, GSH, and 3-MH standards spiked in beer to evaluate the quantitative analysis by these two ion pairs, i.e., ion pair of 130/137 or 218/225. To this end, BQB and BQB-d7 labeled standards were mixed at the concentration ratios of 1:20, 1:10, 1:5, 1:1, 5:1, 10:1, and then the mixture was measured by DPIS using the ion pair of 130/137 or 218/225. The calibration curves were constructed by plotting mean peak intensity ratios of light/heavy versus the mean concentration ratios of light/heavy based on data obtained from triplicate

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RESULTS AND DISCUSSION Principle of the IL−LC−DPIS-MS Strategy. In this IL− LC−DPIS-MS strategy, a pair of isotope labeling reagents (BQB and BQB-d7) containing a reactive group, an isotopically labeled moiety, and an ionizable group were used to label thiolcontaining compounds in beer and human urine. Then BQB and BQB-d7 labeled sample solutions were mixed (1/1, v/v) and detected by LC−DPIS-MS. Two characteristic product ions (m/z 218 and 225 containing the isotope tag) were generated from the BQB and BQB-d7 labeled thiol-containing compounds, respectively. In this respect, two PIS (m/z 218 and 225) could be carried out simultaneously in the MS analysis to record the signals of BQB and BQB-d7 labeled thiol-containing compounds. The advantages of this IL−LC−DPIS-MS strategy includes (1) BQB labeling can significantly enhance the ESI efficiency, which therefore leads to the enhanced detection sensitivity of thiol-containing compounds; moreover, the detection sensitivity also can be further improved by the elevated selectivity due to PIS; (2) BQB and BQB-d7 isotope labeling facilitates the identification of analytes among multiple MS spectral features by selecting the peak pair with characteristic mass difference; (3) with DPIS analysis, BQB and BQB-d7 labeled thiolcontaining compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiolcontaining compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, which can offer accurate identification and quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Some other thiol-derivatizing reagents, such as sulfhydrylreactive reagents, 2-bromo-4′-chloroacetophenone (p-CPB),24 2-bromo-4′-bromoacetophenone (p-BPB),24 isopropylchloroformate (IPCF),25 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate (GITC),26 p-bromo-phenacyl-bromide (p-BMP),27 Ellman’s reagent,28 and popylchloroformate (IPCF)29 were used to derivatize thiols and disulfides followed by analysis using LC−MS. The alkylation of −SH and the introduction of an aromatic group reduce the polarity of the target analytes, enabling a good retention on a reversed-phase column, whereas the fragment ions of these derivatives were complex, and it is difficult to obtain the common characteristic fragment ions for untargeted profiling of thiol-containing compounds using precursor ion scanning. N-(2-Ferroceneethyl)maleimide (FEM) and ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) also has been synthesized to derivatize thiols.30,31 The fragmentation behavior of the FEM and FMEA derivatives (FEM derivatives can generate neutral loss of 66 amu; FMEA can generate one common characteristic fragment ion of m/z 213) enables the detection of unknown thiols by neutral loss scan or precursor ion scan of derivatives. But the synthesis of FEM and FMEA is tedious, and it is also difficult to obtain the isotopic FEM and 9768

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Figure 3. BQB and BQB-d7 labeled beer analyzed by LC−DPIS-MS. (A) Total ion chromatogram of DPIS using the ion pair of 218 and 225. (B) Extracted ion chromatograms by m/z 303 and 310 from BQB and BQB-d7 labeled ion chromatograms, respectively. (C) Extracted ion chromatograms by m/z 462 and 469 from BQB and BQB-d7 labeled ion chromatograms, respectively. (D) Extracted ion chromatograms by m/z 439 and 446 from BQB and BQB-d7 labeled ion chromatograms, respectively.

Table 1. Measured m/z of BQB and BQB-d7 Labeled Thiol-Containing Compounds from Beer and Their Prospective Molecular Formulas Obtained by QTOF Analysis no.

T/min

BQB-labeled (m/z)

BQB-d7-labeled (m/z)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

16.1 8.6 16.5 14.4 23.5 17.9 18.6 15.1 16.7 17.9 27.1 16.5 17.1 24.2 14.8 15.6 17.6 20.8 13.0 19.5 14.0

347.1083 491.1597 303.1138 333.0876 375.0989 377.1142 384.1394 407.1305 407.1283 375.0922 413.1270 416.1240 434.1410 441.2030 449.1310 449.1354 449.1306 462.1980 467.1409 513.2011 524.1739

354.1452 498.2001 310.1502 340.1285 382.1432 384.1578 391.1749 414.1729 414.1733 382.1356 420.1610 423.1658 441.1795 448.1787 456.1749 456.1762 456.1771 469.2301 474.1853 520.2313 531.2150

prospective formulas C5H9NO3S C10H17N3O6S C4H9NOS C4H7NO3S C6H9NO4S C6H11NO4S C8H12N2O2S C7H13NO5S C7H13NO5S C8H5N3OS C8H11N3O3S C13H13PS C9H18N2S3 C13H23NS2 C10H12N5PS C13H15NOS2 C8H15N3O3S2 C10H22N4OS2 C9H18NO5PS C22H19NS C16H20O6S

(163.1910) (307.3210) (119.1820) (149.1640) (191.2010) (193.2170) (200.2560) (223.2430) (223.2430) (191.2098) (229.2562) (232.2811) (250.4476) (257.4584) (265.2745) (265.3943) (265.3530) (278.4379) (283.2817) (329.4580) (340.3914)

measurements. As shown in Supporting Information Figure S3A, the slopes of linear regressions of GSH, Nac, and 3-MH were 0.37, 0.52, and 0.80 by DPIS using the ion pair of 130/ 137, suggesting the ratios of the chromatographic peak intensity inadequately matched with the concentration ratios of the

compound names N-acetylcysteine glutathione 3-aminobutanethioic S-acid formylcysteine (Z)-3-((carboxymethyl)amino)-2-(mercaptomethyl)acrylic acid 2-((2-hydroxyethyl)amino)-3-mercapto-4-oxobutanoic acid (2Z,3Z)-2-(aminomethylene)-N-ethyl-3-formyl-4-mercaptobut-3-enamide 3-((1-carboxy-2-mercaptoethyl)amino)-2-(hydroxymethyl)propanoic acid 4-((1-carboxy-2-mercaptoethyl)amino)-2-hydroxybutanoic acid

different isotope-labeled analytes, whereas the slopes of linear regressions of GSH, Nac, and 3-MH were 0.88, 0.94, 0.91, which were approximate to 1.00 by DPIS using the ion pair of 218/225 (Supporting Information Figure S3B), indicating the ratios of the chromatographic peak area closely matched with 9769

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Figure S5). These results demonstrated that the LC−DPIS-MS method is reliable to profile thiol-containing compounds. We then further examined the 21 thiol-containing compounds by QTOF-MS analysis. The detected peak was analyzed according to ion pair recognition and retention time information from the DPIS. A major advantage of the stable isotope labeling method is that the target analytes can be easily identified by extracting the peak-pair data even if the retention time changed due to the use of a different chromatography column or analytical instrument. The accurate molecular weight of BQB and BQB-d7 labeled thiol-containing compounds and the 21 prospective molecular formulas of the thiol-containing compounds determined using Bruker Daltonics data analysis 4.0 software are shown in Table 1. Beer is a fermented beverage, and the thiol-containing compounds were normally considered as the metabolites of yeast.32 We then searched the molecular formulas of the thiol-containing compounds in the METLIN database, and compounds 1 (C5H9NO3S) and 2 (C10H17N3O6S) were identified to be Nac and GSH. The retention times (Supporting Information Figure S6) and MS/ MS spectra (Supporting Information Figure S7) of BQB and BQB-d7 labeled compounds 1 and 2 from beer are consistent with the standards Nac and GSH spiked in beer (GSH, 1 μmol/L; Nac, 0.05 μmol/L) in the DPIS analysis, which further supports the identified compounds 1 and 2. It is worth noting that Nac existence in beer was first reported by our method. As for the other thiol-containing compounds, their formulas were not found in the METLIN database, indicating they could be some new compounds. And we further determined additional seven thiol-containing compounds in beer (Table 1, compounds 3−9) by their prospective molecular formulas and MS/ MS spectra analysis (Supporting Information Figure S8). Method Validation. We investigated the detection limits of labeled thiol standards (GSH, Nac, and 3-MH) under LC− DPIS−MS conditions. The results showed that the detection limits (calculated at a signal-to-noise ratio of 3) were found to be 37.5, 2.3, and 22.5 nmol/L for GSH, Nac, and 3-MH, respectively. To evaluate the accuracy of the relative quantification of the method, BQB and BQB-d7 labeled beer samples were mixed at different volume ratios (1:10, 1:5, 1:2, 1:1, 2:1, 5:1, and 10:1). The samples were analyzed using LC−DPIS-MS with triplicate measurements. Fifteen BQB and BQB-d7 labeled peak pairs with high intensities were extracted, and their ratios were calculated. The average isotopic ratios calculated from each pair of BQB/BQB-d7 labeling thiols were determined to be 0.10, 0.22, 0.51, 1.02, 2.10, 5.11, and 9.72 for the 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, and 10:1 mixtures, respectively. The results show that the chromatographic peak intensity ratios highly matched with the concentration ratios of the different isotope-labeled analytes with relative standard deviations (RSDs) being less than 15.6% (Figure 4). On the basis of the peak area ratios of BQB/BQB-d7-labeled derivatives, we can determine the absolute concentration of each metabolite in the samples. BQB-d7-labeled standards were spiked into the BQB-labeled beer sample, and the concentration of relevant compounds can be determined based on the measured peak area ratios of BQB/BQB-d7-labeled derivatives. The GSH and Nac were quantified to be 2.09 and 0.31 μmol/L in beer, respectively. Effect of H 2 O 2 Treatment on Thiol-Containing Compounds in Beer. Thiol-containing compounds are important for the antioxidation capability of beer.33 As reducing

the concentration ratios of the different isotope-labeled analytes. The coefficients of determination (R2) were also found to be close to 1.00, demonstrating good linearities by DPIS using the ion pair of 218/225. The better correlation obtained by DPIS using the ion pair of 218/225 rather than 130/137 may be attributed to that the product ions of 218 and 225 assigned to the fragmentation of the C−S bond in BQB and BQB-d7 derivatives were more specific for the selection of the thiol-containing compounds. Moreover, the product ions of 130 and 137 are at the low-mass region that typically exhibits significant background noise from fragment ions of solvent or contaminants. We also examined the ion pairs of 218 and 225 in the qualitative analysis of the mixed BQB and BQB-d7 labeled standards (1/1, v/v). The results showed that the peak areas of the three thiol standards (GSH, Nac, and 3-MH) were similar, and the isotopic effect was approximate 0.1 min on the reversed-phase LC separation (Supporting Information Figure S4); the slight retention time difference did not influence the identification of thiol-containing compounds by LC−DPIS-MS method. Therefore, the ion pairs of 218 and 225 were used in the subsequent DPIS analysis. Qualitative Analysis of Thiol-Containing Compounds by LC−DPIS-MS in Beer. For the qualitative profiling of thiolcontaining compounds from beer, an equal volume of beer was labeled with BQB or BQB-d7, respectively. Then the light- and heavy-labeled samples were mixed and analyzed by LC−DPISMS. The LC−DPIS-MS method consisted of two PIS of m/z 218 and 225, which generated two individual ion chromatograms corresponding to the precursor ion of BQB and BQB-d 7 labeled thiol-containing compounds, respectively. Peak-pair data were extracted from the two ion chromatograms according to a mass shift of n × 7 Da (i.e., MBQB‑d7‑labeled − MBQB‑labeled = n × 7 Da), and only peak pairs with the same retention time and intensity were assigned to be the candidates of thiol-containing compounds. The structures of the assigned candidates were further elucidated by product ion scan (MS/MS) and highresolution mass spectrometry (QTOF-MS) analysis. Figure 3A shows the total ion chromatograms of beer sample analyzed by DPIS using the ion pair of 218/225. The chromatograms derived from the BQB and BQB-d7 labeled beer samples display almost identical peak patterns. Peak-pair data were extracted from the BQB and BQB-d7 labeled chromatograms according to the aforementioned criteria, and 21 ion pairs were detected (Table 1). Taking compound 1 and 17 as the examples, the peak intensities and retention times were the same in the extracted ion chromatograms at m/z 303/ 310 or m/z 462/469 from BQB and BQB-d7 labeled samples (Figure 3, parts B and C), suggesting these candidate compounds could be thiol-containing compounds. On the contrary, in the extracted ion chromatograms at m/z 439 and 446, the peak was only found in the BQB-labeled sample and no peak was observed in BQB-d7-labeled sample, indicating that this compound should not be a thiol-containing compound (Figure 3D). For the MS/MS analysis, similar to the thiol standards (GSH, Nac, and 3-MH), all the BQB-labeled thiol candidates possess two common product ions of m/z 130 and 218, and BQB-d7labeled thiol candidates possess two common product ions of m/z 137 and 225, indicating that all the 21 thiol candidates were thiol-containing compounds (Supporting Information 9770

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mode under the same chromatography conditions. Compared to full scan mode (Figure 6A), DPIS has two major advantages. First, DPIS generates two individual ion chromatograms corresponding to BQB and BQB-d7 labeled samples (Figure 6B). In this respect, the analytes from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, which can eliminate the MS response fluctuation and mutual interference. The ion signals between the light- and heavy-labeled samples can be clearly distinguished; therefore, the accuracy to select the peakpair data from the light- and heavy-labeled sample chromatograms was significantly improved, whereas only one chromatogram was obtained under full scan mode, so the mutual interference from the light- and heavy-labeled ion signals was inevitable. In fact, it is difficult to select the peak-pairs using either low-resolution (QTRAP) or high-resolution (QTOF) mass spectrometry under full scan mode. Second, DPIS monitors the characteristic ion fragment produced by the fragmentation of precursor ions, which can effectively eliminate the interference from the unlabeled compounds or other fragments. Thus, DPIS can significantly improve the detection selectivity and sensitivity. Figure 6C shows the extracted ion chromatograms of BQB and BQB-d7 labeled Nac at the m/z 347 (S/N, 14) and 354 (S/N, 16) in beer under full scan mode, which have strong background noise signals. Nevertheless, the extracted ion chromatograms obtained under DPIS mode are much clearer, and the S/N was 116 and 130 for the BQB and BQB-d7 labeled Nac, respectively (Figure 6D). The same results was also observed in MS spectra (Figure 6, parts E and F). Taken together, the DPIS method can significantly improve the detection accuracy, selectivity, and sensitivity, and the distinctly identified quasimolecular ions of the thiol-containing compounds are beneficial to further elucidate their structures. Profiling of Thiol-Containing Compounds by LC− DPIS-MS in Human Urine. To extend the LC−DPIS-MS method to more challenging biological samples, we further profiled thiol-containing compounds in human urine. The results showed that 103 thiol candidates in urine were identified (Supporting Information Table S2). Their structures were further elucidated by MS/MS and high-resolution mass spectrometry analysis. Among the 103 thiol candidates, 66 had distinct MS/MS spectra. Through the comparison of retention times (Supporting Information Figure S10) and MS/ MS spectra with the standards, compounds 1, 2, 3, 4, and 5 from human urine were identified to be Cys, Hcys, Nac, γ-GluCys, and GSH with concentrations of 12.3, 3.06, 1.92, 0.13, 0.68 μmol/L, respectively. Another 12 thiol candidates were identified by their prospective molecular formulas and MS/MS spectra (Supporting Information Table S2, compounds 6−17). The proposed compound structures and fragmentation pathways are shown in Supporting Information Figure S11. It was worth noting that Cys, Hcys, Nac, γ-Glu-Cys, and GSH have been previously reported in human urine,23,34 while the other thiol-containing compounds were first identified in human urine in the current study.

Figure 4. Regression line of the measured peak intensity ratios vs the mean concentration ratios (1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1) of 15 BQB/BQB-d7-labeled peak pairs with high intensities from beer.

agents, thiol-containing compounds could be oxidized upon H2O2 treatment. In this respect, we used a forward and reverse labeling strategy to examine the contents change of the thiolcontaining compounds in beer by H2O2 treatment. The measured peak intensity ratios of treated/untreated of 21 thiol-containing compounds in the beer are shown in the Supporting Information Table S1. As described in Figure 5, the

Figure 5. Effect of 1% H2O2 treatment on the contents change of 21 thiol-containing compounds in beer. Forward labeling, the untreated beer sample was labeled with BQB and the treated sample was labeled by BQB-d7. Reversed labeling, the untreated beer sample was labeled with BQB-d7 and the treated sample was labeled by BQB.

results obtained by forward labeling and reversed labeling were similar. And all the ratios of treated/untreated were less than 1, demonstrating that thiol-containing compounds were reduced after the H2O2 treatment. We also performed the studies at three different concentrations of H2O2 (0.5%, 1%, 2%) with different treatment time periods (1 and 2 h) (Supporting Information Figure S9). The results showed that the contents of the thiol-containing compounds decreased as the concentration of H2O2 and treatment time increased. However, even at the condition of 2% H2O2 treatment with 2 h, it was difficult to oxidize all the thiol-containing compounds. We reason that the insufficient oxidation of thiol-containing compounds may be due to the existence of other reduction reagents in beer. Comparison of Precursor Ion Scan with Full Scan Method. The BQB and BQB-d7 labeled beer sample (1/1, v/ v) was analyzed by LC−MS with either full scan mode or DPIS

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CONCLUSION In the current study, we developed an IL−LC−DPIS-MS method for the profiling of thiol-containing compounds in beer and human urine. Two characteristic product ions m/z 218 and 225 that contain an isotope tag were generated from the BQB and BQB-d7 labeled compounds, respectively, which were used 9771

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Figure 6. Comparison of the DPIS with full scan mode. (A) Total ion chromatogram obtained under full scan mode. (B) Total ion chromatogram obtained under DPIS mode. (C) Extracted ion chromatograms of BQB and BQB-d7 labeled Nac at m/z 347 and 354 from beer under full scan mode. (D) Extracted ion chromatograms of BQB and BQB-d7 labeled Nac at m/z 347 and 354 from beer under DPIS mode. (E) MS spectrum of BQB and BQB-d7 labeled Nac from beer under full scan mode. (F) Mass spectrum of BQB and BQB-d7 labeled Nac from beer under DPIS mode.

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ACKNOWLEDGMENTS The authors are thankful for the financial support from the National Natural Science Foundation of China (21475098, 91217309, 91017013), and we thank Mr. Shu-Jian Zheng for the help on the fragmentation pathway analysis for the identified compounds.

for DPIS in MS analysis. The method significantly improved the detection accuracy, selectivity, and sensitivity in the analysis of thiol-containing compounds. Using this method, 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with standards or tandem mass spectrometry analysis. This ILDPIS-MS method can also be extended for the efficient identification and quantification of a relatively wide group of metabolites that contain an identical group in metabolomics study.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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Corresponding Authors

*Phone: +86-27-68755595. Fax: +86-27-68755595. E-mail: [email protected]. *Phone: +86-27-68755595. Fax: +86-27-68755595. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 9772

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