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Multiplexed Quantification of Metabolites with MISSILE

Jones DR1*, Wang X2, Shaw T2,3, Cho JH2 and Peng J2-5*

1Department of Biochemistry and Molecular Pharmacology, Langone Medical Center, New York University, NY, 10016, USA

2St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN 38105, USA

3Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA

4Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA

5Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA

*Corresponding Author:
Jones DR
Department of Biochemistry and Molecular Pharmacology
Langone Medical Center
New York University
550 1st Avenue, NY, 10016, USA
Tel: 646-501-2097
E-mail: [email protected]
Peng J
Department of Structural Biology
St. Jude Children's Research Hospital
262 Danny Thomas Place, Memphis, TN 38105, USA
Tel: (901) 595-7499
Fax: (901) 595-3032
E-mail: [email protected]

Received Date: February 06, 2017; Accepted Date: April 06, 2017; Published Date: April 12, 2017

Citation: Jones DR, Wang X, Shaw T, Cho JH, Peng J (2017) Multiplexed Quantification of Metabolites with MISSILE. Metabolomics (Los Angel). 7:189. doi: 10.4172/2153-0769.1000189

Copyright: © 2017 Jones DR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

We tested a strategy for multiplexed (4-plex) quantification of metabolites using the MISSILE identification method with liquid chromatography coupled to tandem mass spectrometry. We applied this methodology to study the metabolic effect of the proteasome inhibitor and chemotherapeutic drug Bortezomib in yeast cells. Using JUMPm software version 1.1 we simultaneously identified and quantified 95 metabolites across four experimental conditions and found that Bortezomib increased the accumulation of dipeptides but decreased the levels of specific lipid molecules (e.g. phosphtidylethanolamines) in a dose-dependent manner. This method combines metabolite identification and quantification, making untargeted metabolomics experiments more informative.

Keywords

Metabolomics; Metabolome; Mass spectrometry; Stable isotope labeling; Liquid chromatography; Metabolite quantification; Multiplex; Bortezomib; Proteasome inhibition; MISSILE

Abbreviations

MISSILE: Metabolome Identification by Systematic Stable Isotope Labeling Experiments; nUPLC-HRMS: Nanoscale Ultra-Performance Liquid Chromatography–High Resolution Mass Spectrometry

Introduction

We recently described a formula-centric strategy for the identification of metabolites in untargeted metabolomics experiments [1,2]. The method uses stable isotope labeling [3-5] to identify the formula and structure of metabolites in an automated fashion with support for false discovery rate estimation [6,7]. Here we have expanded on this strategy by adding a fourth isotope labeling condition to enable 4-plex relative quantification of metabolites in LC-MS experiments. Each label represents an independent experimental condition so that we can compare the relative levels of metabolites from pairwise duplicates, time course, or dose-response experimental designs. Multi-plexed analyses offer many advantages over traditional label-free methods [8], but current strategies rely on specific functional group tags to chemically label metabolites [9] which limit the scope of quantified metabolites and introduce complexity to the sample preparation workflow. As a test of our methodology, we performed a dose-response analysis on the effect of the proteasome inhibitor Bortezomib [10] in yeast cells. This drug is an effective therapy for multiple-myeloma, but drug-resistance invariably develops due to incompletely characterized mechanisms [11,12]. The effect of proteasome inhibition on protein [13] and gene regulation [14] has been studied in a variety of contexts, but much less is known about the role of metabolism in this process. We report our preliminary investigation and demonstrate the analytical tools for carrying out multiplexed metabolomics analyses.

Methods

Materials–LC-MS grade acetonitrile (ACN), water, and formic acid (Sigma), glass beads (Next Advance), Difco™ yeast nitrogen base, DMEM (BD Biosciences), nanoLC column (Waters), 13C-6 glucose, 15N-2 ammonium sulfate, (Cambridge Isotope Laboratories).

MISSILE protocol

For Saccharomyces cerevisiae (Fleischmann) labeling, cells were grown in four different minimal media conditions. A control media consisted of natural isotopic abundance components; Difco™ yeast nitrogen base without amino acids and ammonium sulfate (BD Biosciences), with 5 g/L ammonium sulfate (Sigma), and 20 g/L glucose (Sigma). For carbon-13 labeling, the media remained the same except that 13C-6 glucose (Cambridge Isotope Laboratories) was used in place of standard glucose. Similarly for nitrogen-15 labeling, 15N-2 ammonium sulfate was substituted into the media. Each culture was maintained for ~30 generations in the labeled media before drug treatment and metabolite extraction. For Bortezomib treatment, cultures were seeded to an OD600 of 0.1 and allowed to grow to 1 (~6 generations).

Bortezomib treatment

Bortezomib was solubilized in 10 mM, 1 mM, and 0.1 mM stock solutions (water) so that equal volumes could be spiked in for each treatment. For each 25 mL yeast culture, the OD600 was monitored and recorded every hour. Bortezomib was added to each culture at OD600=0.5 absorbance units.

Sample preparation

Control and drug treatment yeast cultures were harvested and their metabolites were extracted. The liquid cultures were transferred to 15 mL conical vials and centrifuged at 1,000 g for 3 min to obtain a cell pellet. The supernatant was discarded and 1 mL of freezing 80% acetonitrile was added. Each vial was subjected to 3 min of vortexing at 3,000 rpm in a 1 on/1 off pattern to maintain sample temperature [2]. The lysate was transferred to a fresh vial to exclude the glass beads, and then centrifuged at 21,000 g for 5 min to clarify the liquid phase.

Spectrophotometric determination of multiplex mixing ratio

The absorption (300 nm) of each supernatant was measured in a 1 mL cuvette and used to calculate the mixing ratio of the 4 individual cultures. For each culture we multiplied the absorbance reading by the volume of supernatant recovered (900 μL) as an estimate of the total amount of metabolites present. We then calculated the amount of each sample needed to equal 90% of the lowest measured label. Using these values we mixed the supernatants of the 4 labeled samples into a single tube (~3 mL), vortexed, and aliquoted the mixture into 4 separate tubes.

Sample reconstitution

The mixed or individual aliquots were dried under centrifugal vacuum and resolubilized to 75 μL in buffer A and transferred to inserts for LC-MS analysis.

LC-MS analysis and parameters

Bortezomib treated yeast samples were analyzed on an Orbitrap Elite (Thermo Scientific) coupled to an Easy nLC™ system as previously described [2]. In summary, we used a nano Acquity UPLC column (75 μm × 100 mm) packed with 1.7 μm BEH C18 beads with 0.2% formic acid in water (Mobile Phase A) or acetonitrile (Mobile Phase B). Sample injection volume was 2 μL. LC-MS analysis was performed in positive ion (3 kV) mode with a 15 μm, 5 cm PicoTip emitter (New Objective). A top 5 data-dependent method was used to target ions for fragmentation (MS/MS) for later structural identification.

Results and Discussion

Proteasome inhibition of yeast cells and LC-MS analysis

Yeast cells in a respiring liquid culture were subjected to proteasome inhibition by addition of Bortezomib. We treated cells with three concentrations of drug; at the reported IC50 [15], 10-fold below, and 10-fold above, with a no-drug control (Figure 1a). To distinguish these four experimental conditions, carbon (i.e., glucose) and nitrogen (ammonium sulfate) sources in the culture were exchanged for various heavy stable isotope labeled compounds (i.e., 13C-6-glucose and 15N-ammonium sulfate). There was a decrease in the yeast growth rate with increasing concentrations of Bortezomib (Figure 1b). We subjected each condition to LC-MS analysis alone (Figure 1c) or multiplexed (mixed together) and observed changes in the base-peak chromatogram due to drug-treatment.

metabolomics-identified-metabolites

Figure 1: Metabolomics analysis of Bortezomib treated yeast cells (a) Experimental design of the 4-plex study. Labels indicate the stable isotope culture condition. Condition 4 (red) used both stable isotope labels simultaneously. (b) Growth curve for the four independent yeast cultures, monitored by OD600. (c) Base-peak chromatograms for the four cultures analyzed independently prior to multiplex analysis. (d) An example MS1 spectrum for a metabolite peak after mixing the four yeast samples together. Colored peaks reflect the increased mass of the metabolite from stable isotope labelling and also the relative intensity changes due to Bortezomib treatment. This metabolite was identified as PE (15:0/14:1(9Z)). (e) Automated chemical formula-determination workflow for the example metabolite peaks shown in (d). Candidates represent chemical formulas within 4 ppm mass tolerance of the unlabelled peak. With increasingly strict search criteria the number of possible formulas dramatically decreases. The determined formula represents the charged parent ion. All formulas in Table 1 have been discharged. (f) Extracted ion chromatogram for each of the four labeled versions of the yeast metabolite shown in (d). Retention time and peak shape are not affected by isotope labelling, while the peak intensity reflects the changes due to Bortezomib treatment. (g) Tandem mass spectrum (MS/MS) of the labeled precursors from the example metabolite. (d-f) Each structure fragment shows two peaks, representing the two isotopes labeled parent ions which were fragmented in the Top 5 data-dependent method. (h) Fold change distribution of quantified metabolites compared to the no-drug control. Higher Bortezomib concentrations were associated with increased variation in metabolite levels. (i) Summary of JUMPm global metabolite search results. The formula false discovery rate (FDR) was less than 1% as determined by JUMPm based on the relative frequency of target and decoy formulas detected. (j-l) Up-regulated, unaffected, and down-regulated metabolites respectively as a function of Bortezomib concentration. Labels indicate identified metabolites using JUMPm software.

Formula and structure identification of Bortezomib regulated metabolites

We used JUMPm software to globally analyze the metabolites from yeast cells treated with Bortezomib. As an example, we manually examined one of the down-regulated metabolites (a phosphatidylethanolamine). The MS1 scan shows four ions of varying mass, reflecting total incorporation of heavy stable isotopes into the chemical structure (Figure 1d). The mass shift of the four ions can also be used to determine the chemical formula of the metabolite (Figure 1e) as previously described. Each of the four parent ions for phosphatidylethanolamine was manually extracted to confirm their co-elution and identity as isotope labels (Figure 1f). The intensity of these peaks was detected by JUMPm and serves as the basis for the relative quantification. The structure identity was determined by JUMPm in a search of its associated MS/MS spectra (Figure 1g). These tandem mass spectra were collected in a Top 5 data-dependent fashion. For phosphatidylethanolamine, MS2 spectra were acquired from two of the four labeled parent ions, the 12C parent (unlabeled control) and the double labeled 13C15N parent ion. Two structural fragments were observed for each parent with nearly identical relative intensities. Overall, we identified 181 metabolite formulas and quantified 95 metabolite structures among the four treatment conditions with a false discovery rate of less than 1% (Table 1).

MS1 scan 12C m/z 15N m/z 13C m/z 13C/15N m/z 12C Intensity 15N Intensity 13C Intensity 13C/15N Intensity Formula MS2 scan Log2 Fold Change      (0.3µM/Con)  Name SMILES InChIKey Mscore Precursor Label Type MS1      S/N
7087 480.3097 481.3062 503.387 504.384 1721102 503870 247540 406383 C23H46NO7P 7048 -2.8 LysoPE(18:1(9Z)/0:0) [H][[email protected]@](O)(COC(=O)CCCCCCC\C=C/CCCCCCCC)COP(O)(=O)OCCN PYVRVRFVLRNJLY-MZMPXXGTSA-N 22.2 N15 29
7087 522.3567 523.3535 548.4441 549.441 7158426 1098587 1090479 1319713 C26H52NO7P 7116 -2.71 LysoPC(18:1(11Z)) CCCCCCC=CCCCCCCCCCC(=O)OC[[email protected]@H](O)COP([O-])(=O)OCC[N+](C)(C)C PZRFVAHZNWPPAC-RUZDIDTESA-N 0 C13 95
7087 648.4615 649.4606 682.5757 683.5724 2451481 1121747 498976 595524 C34H66NO8P 7095 -2.3 PE(15:0/14:1(9Z)) [H][[email protected]@](COC(=O)CCCCCCCCCCCCCC)(COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCC OIERHHCSSSRIJH-PGKKXZESSA-N 60.7 N15 43
5991 452.2785 453.2765 473.3489 474.3459 6712169 6533236 1728196 2895832 C21H42NO7P 6021 -1.96 LysoPE(16:1(9Z)/0:0) [H][[email protected]@](O)(COC(=O)CCCCCCC\C=C/CCCCCC)COP(O)(=O)OCCN DSOWUEHXZJUNID-WHXUGTBJSA-N 13.9 N15 1026
6194 298.2749 299.2719 316.3353 317.3323 529804 277625 207786 171710 C18H35NO2 6190 -1.35 Palmitoleoyl Ethanolamide CCCCCC\C=C/CCCCCCCC(=O)NCCO WFRLANWAASSSFV-FPLPWBNLSA-N 0 C13 52
348 130.0503 131.0474 135.0672 136.0642 8262 6279 3432 10685 C5H7NO3 329 -1.27 1-Pyrroline-4-hydroxy-2-carboxylate OC1CN=C(C1)C(O)=O AOMLMYXPXUTBQH-UHFFFAOYSA-N 8.5 C13 68
273 385.1306 391.1131 399.1777 405.1599 3266 1017 1842 2050 C14H20N6O5S 266 -0.83 S-Adenosylhomocysteine N[[email protected]@H](CCSC[[email protected]]1O[[email protected]]([[email protected]](O)[[email protected]@H]1O)N1C=NC2=C(N)N=CN=C12)C(O)=O ZJUKTBDSGOFHSH-WFMPWKQPSA-N 4.5 N15 12
5138 440.2786 441.2757 460.3458 461.3429 515593 650568 308222 319693 C20H42NO7P 5093 -0.74 LysoPE(0:0/15:0) [H][[email protected]@](CO)(COP(O)(=O)OCCN)OC(=O)CCCCCCCCCCCCCC GIUNBNKDUHWVHI-LJQANCHMSA-N 32.9 C12 80
651 104.1071 105.1042 109.1239 110.121 122152 114269 75907 126557 C5H13NO 671 -0.69 Neurine [OH-].C[N+](C)(C)C=C NIPLIJLVGZCKMP-UHFFFAOYSA-M 0 N15 23
256 166.087 167.084 175.1173 176.1142 1592 1494 1028 3138 C9H11NO2 275 -0.63 Methyl N-methylanthranilate CNC1=CC=CC=C1C(=O)OC GVOWHGSUZUUUDR-UHFFFAOYSA-N 0 N15 22
259 148.061 149.0581 153.0778 154.0748 5494 4859 3627 16905 C5H9NO4 260 -0.6 DL-Glutamate NC(CCC(O)=O)C(O)=O WHUUTDBJXJRKMK-UHFFFAOYNA-N 0 C12 92
516 118.0865 119.0836 123.1034 124.1004 70584 66231 48595 90671 C5H11NO2 483 -0.54 Norvaline CCC[[email protected]@H](N)C(O)=O SNDPXSYFESPGGJ-SCSAIBSYSA-N 2.7 C13 29
348 159.0771 161.0712 165.0972 167.0913 9222 5738 7129 9637 C6H10N2O3 338 -0.37 1-(Hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione CC1(C)N(CO)C(=O)NC1=O SIQZJFKTROUNPI-UHFFFAOYSA-N 2.6 C12 62
309 148.061 149.058 153.0778 154.0748 4897 5281 3834 8654 C5H9NO4 260 -0.35 DL-Glutamate NC(CCC(O)=O)C(O)=O WHUUTDBJXJRKMK-UHFFFAOYNA-N 0 C12 56
7039 118.0864 119.0835 123.1032 124.1003 232026 227382 183446 304186 C5H11NO2 6994 -0.34 Norvaline CCC[[email protected]@H](N)C(O)=O SNDPXSYFESPGGJ-SCSAIBSYSA-N 0 C13 41
7159 159.0768 161.0709 165.097 167.091 541456 352087 444023 532270 C6H10N2O3 7150 -0.29 1-(Hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione CC1(C)N(CO)C(=O)NC1=O SIQZJFKTROUNPI-UHFFFAOYSA-N 0 C12 54
7153 176.1034 179.0946 182.1236 185.1147 1117889 694709 941790 1316654 C6H13N3O3 7142 -0.25 Citrulline N[[email protected]@H](CCCNC(N)=O)C(O)=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0 C12 118
7911 148.0609 149.058 153.0777 154.0747 294553 319728 252496 469843 C5H9NO4 7931 -0.22 D-Glutamic acid N[[email protected]](CCC(O)=O)C(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0 C12 782
720 176.1035 179.0948 182.1237 185.1151 742079 525381 641098 810360 C6H13N3O3 698 -0.21 Citrulline N[[email protected]@H](CCCNC(N)=O)C(O)=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0 C12 180
1617 298.0975 303.0827 309.1344 314.1196 17453480 16726398 15118440 18394530 C11H15N5O3S 1618 -0.21 5'-Methylthioadenosine CSC[[email protected]]1O[[email protected]]([[email protected]](O)[[email protected]@H]1O)N1C=NC2=C1N=CN=C2N WUUGFSXJNOTRMR-IOSLPCCCSA-N 5.1 C12 570
145 176.1038 179.0949 182.1239 185.1151 5172 2300 4496 4730 C6H13N3O3 141 -0.2 Argininic acid NC(=N)NCCC[[email protected]](O)C(O)=O BMFMQGXDDJALKQ-BYPYZUCNSA-N 11.2 C12 30
309 166.0869 167.0839 175.1172 176.1142 1076 953 938 1889 C9H11NO2 275 -0.2 D-Phenylalanine C1=CC=C(C=C1)CC(C(=O)[O-])[NH3+] COLNVLDHVKWLRT-MRVPVSSYSA-N 0 N15 13
516 104.1072 105.1042 109.124 110.121 17570 14112 15487 10017 C5H13NO 507 -0.18 Neurine [OH-].C[N+](C)(C)C=C NIPLIJLVGZCKMP-UHFFFAOYSA-M 0 C13 4
250 188.0712 189.0682 199.1082 200.1058 11300 10478 10192 32140 C11H9NO2 251 -0.15 Indoleacrylic acid OC(=O)\C=C\C1=CC2=C(N1)C=CC=C2 SXOUIMVOMIGLHO-AATRIKPKSA-N 11.6 C12 194
268 263.1402 265.1344 277.1872 279.1814 2243 1315 2075 1785 C14H18N2O3 222 -0.11 L-phenylalanyl-L-proline NC(CC1=CC=CC=C1)C(=O)N1CCCC1C(O)=O WEQJQNWXCSUVMA-UHFFFAOYSA-N 46 C13 10
382 118.0866 119.0837 123.1034 124.1006 1712 2507 1679 8063 C5H11NO2 401 -0.03 L-Valine CC(C)[[email protected]](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 3.1 C13 47
145 148.0611 149.0581 153.0779 154.0749 3782 4585 3738 6096 C5H9NO4 164 -0.02 N-Acetyl-DL-serine CC(=O)NC(CO)C(=O)O JJIHLJJYMXLCOY-UHFFFAOYSA-N 7.2 C13 38
264 166.0869 167.084 175.1173 176.1142 3501 3232 3601 3138 C9H11NO2 275 0.04 Methyl N-methylanthranilate CNC1=CC=CC=C1C(=O)OC GVOWHGSUZUUUDR-UHFFFAOYSA-N 0 N15 22
273 152.0575 157.0426 157.0742 162.0593 5610 5698 5851 3301 C5H5N5O 235 0.06 Guanine NC1=NC(=O)C2=C(N1)N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 4 C13 20
180 159.0771 161.0712 165.0973 167.0915 1844 771 1961 653 C6H10N2O3 223 0.09 1-(Hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione CC1(C)N(CO)C(=O)NC1=O SIQZJFKTROUNPI-UHFFFAOYSA-N 0 N15 4
284 205.098 207.0921 216.135 218.1291 5375 6216 5723 2568 C11H12N2O2 265 0.09 (±)-Tryptophan NC(CC1=CNC2=CC=CC=C12)C(O)=O QIVBCDIJIAJPQS-UHFFFAOYNA-N 17 N15 17
504 188.0713 189.0683 199.1082 200.1053 292778 278202 313539 430796 C11H9NO2 456 0.1 Indoleacrylic acid OC(=O)\C=C\C1=CC2=C(N1)C=CC=C2 SXOUIMVOMIGLHO-AATRIKPKSA-N 0 C12 137
273 136.0624 141.0476 141.0792 146.0642 3002 1781 3233 1467 C5H5N5 277 0.11 Adenine NC1=NC=NC2=C1NC=N2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0 C13 9
264 116.071 117.068 121.0878 122.0848 1509 1096 1632 557 C5H9NO2 274 0.11 4-Amino-2-methylenebutanoic acid NCCC(=C)C(O)=O FTWHFXMUJQRNBK-UHFFFAOYSA-N 4 C13 4
497 210.0768 211.0739 220.1104 221.1074 31546 48871 34809 75814 C10H11NO4 511 0.14 N-Formyl-L-tyrosine C1=CC(=CC=C1CC(C(=O)O)NC=O)O ROUWPHMRHBMAFE-VIFPVBQESA-N 0 C13 24
1929 457.1132 461.1014 474.1702 478.1584 777329 845863 865254 1128229 C17H21N4O9P 1891 0.15 Flavin Mononucleotide CC1=CC2=C(C=C1C)N(C[[email protected]](O)[[email protected]](O)[[email protected]](O)COP(O)(O)=O)C1=NC(=O)NC(=O)C1=N2 FVTCRASFADXXNN-SCRDCRAPSA-N 0 C12 69
301 156.0774 159.0684 162.0975 165.0887 1322 1265 1485 2515 C6H9N3O2 338 0.17 L-2-Amino-3-(1-pyrazolyl)propanoic acid NC(CN1C=CC=N1)C(O)=O PIGOPELHGLPKLL-UHFFFAOYNA-N 5 N15 17
497 348.0715 353.0566 358.1052 363.0902 25113 32106 28228 56363 C10H14N5O7P 533 0.17 2-hydroxy-dAMP [H][[email protected]]1(O)C[[email protected]@]([H])(O[[email protected]]1([H])COP(O)(O)=O)N1C=NC2=C(N)N=C(O)N=C12 GEQDRKVFKBSPSW-KVQBGUIXSA-N 11.2 N15 17
259 159.0771 161.0711 165.0972 167.0914 782 743 880 9293 C6H10N2O3 223 0.17 1-(Hydroxymethyl)-5,5-dimethyl-2,4-imidazolidinedione CC1(C)N(CO)C(=O)NC1=O SIQZJFKTROUNPI-UHFFFAOYSA-N 0 N15 56
273 188.0715 189.0685 199.1085 200.1054 20843 23582 23623 14358 C11H9NO2 251 0.18 Indoleacrylic acid OC(=O)\C=C\C1=CC2=C(N1)C=CC=C2 SXOUIMVOMIGLHO-AATRIKPKSA-N 11.6 C12 88
2929 318.182 321.1732 335.2391 338.2303 712582 415598 812220 568953 C17H23N3O3 2883 0.19 Tryptophyl-Isoleucine CCC(C)C(NC(=O)C(N)CC1=CNC2=C1C=CC=C2)C(O)=O PITVQFJBUFDJDD-UHFFFAOYSA-N 0 C13 65
3421 175.1194 179.1076 181.1396 185.1277 953532 1225147 1091760 1934711 C6H14N4O2 3374 0.2 L-Arginine N[[email protected]@H](CCCN=C(N)N)C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-N 0 C13 252
497 188.0713 189.0683 199.1083 200.1053 185514 190547 212761 430796 C11H9NO2 456 0.2 Indoleacrylic acid OC(=O)\C=C\C1=CC2=C(N1)C=CC=C2 SXOUIMVOMIGLHO-AATRIKPKSA-N 0 C12 137
249 205.0979 207.092 216.1349 218.129 4919 4893 5842 6544 C11H12N2O2 265 0.25 (±)-Tryptophan NC(CC1=CNC2=CC=CC=C12)C(O)=O QIVBCDIJIAJPQS-UHFFFAOYNA-N 17 N15 36
7394 348.0712 353.0564 358.1048 363.0901 33807 41400 40979 61140 C10H14N5O7P 7351 0.28 3'-AMP NC1=C2N=CN([[email protected]@H]3O[[email protected]](CO)[[email protected]@H](OP(O)(O)=O)[[email protected]]3O)C2=NC=N1 LNQVTSROQXJCDD-KQYNXXCUSA-N 8.3 C13 63
238 272.1733 277.1585 283.2103 288.1953 8982 11042 11020 11400 C11H21N5O3 222 0.3 Arginyl-Proline NC(CCCNC(N)=N)C(=O)N1CCCC1C(O)=O LQJAALCCPOTJGB-UHFFFAOYSA-N 62.8 N15 68
273 137.0464 141.0346 142.0632 146.0513 5164 5365 6561 480 C5H4N4O 277 0.35 Hypoxanthine OC1=NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0 C13 3
300 116.0709 117.0679 121.0877 122.0848 929 529 1186 570 C5H9NO2 274 0.35 4-Amino-2-methylenebutanoic acid NCCC(=C)C(O)=O FTWHFXMUJQRNBK-UHFFFAOYSA-N 4 C13 4
5192 583.2573 587.2454 616.3681 620.3563 223013 284064 285828 387537 C33H34N4O6 5199 0.36 Biliverdin CC1=C(C=C)\C(NC1=O)=C\C1=C(C)C(CCC(O)=O)=C(N1)\C=C1/N\C(=C\C2=NC(=O)C(C=C)=C2C)C(C)=C1CCC(O)=O RCNSAJSGRJSBKK-YKSNQIBWSA-N 0 C13 68
7213 348.0713 353.0565 358.1049 363.0901 196288 247135 260942 393713 C10H14N5O7P 7197 0.41 2'-Deoxyguanosine 5'-monophosphate NC1=NC2=C(N=CN2[[email protected]]2C[[email protected]](O)[[email protected]@H](COP(O)(O)=O)O2)C(=O)N1 LTFMZDNNPPEQNG-KVQBGUIXSA-N 29.5 N15 77
7255 175.1195 179.1076 181.1396 185.1277 1477916 1796139 1994965 3215890 C6H14N4O2 7208 0.43 D-Arginine N[[email protected]](CCCNC(N)=N)C(O)=O ODKSFYDXXFIFQN-SCSAIBSYSA-N 0 C12 1052
497 303.1462 307.1344 318.1967 322.1844 28925 30996 39127 43694 C15H18N4O3 480 0.44 Histidinyl-Phenylalanine NC(CC1=CN=CN1)C(=O)NC(CC1=CC=CC=C1)C(O)=O XMAUFHMAAVTODF-UHFFFAOYSA-N 20.1 C12 14
7159 308.0919 311.083 318.1254 321.1165 408645 495074 557197 678460 C10H17N3O6S 7157 0.45 Glutathione N[[email protected]@H](CCC(=O)N[[email protected]@H](CS)C(=O)NCC(O)=O)C(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 19.2 C12 68
378 308.0924 311.0835 318.1259 321.117 8656 11535 11839 13974 C10H17N3O6S 344 0.45 Glutathione N[[email protected]@H](CCC(=O)N[[email protected]@H](CS)C(=O)NCC(O)=O)C(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0 C13 88
759 175.1195 179.1077 181.1396 185.1277 1707376 2330049 2339478 3978705 C6H14N4O2 732 0.45 D-Arginine N[[email protected]](CCCNC(N)=N)C(O)=O ODKSFYDXXFIFQN-SCSAIBSYSA-N 0 C13 1033
7255 147.1132 149.1073 153.1333 155.1274 47481 18895 65506 46668 C6H14N2O2 7220 0.46 D-Lysine NCCCC[[email protected]@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0 C12 13
243 308.0927 311.0838 318.1263 321.1174 20323 27942 28097 32068 C10H17N3O6S 213 0.47 Glutathione N[[email protected]@H](CCC(=O)N[[email protected]@H](CS)C(=O)NCC(O)=O)C(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 11.8 C12 193
378 303.1464 307.1346 318.1969 322.185 1898 1715 2637 2006 C15H18N4O3 344 0.47 Histidinyl-Phenylalanine NC(CC1=CN=CN1)C(=O)NC(CC1=CC=CC=C1)C(O)=O XMAUFHMAAVTODF-UHFFFAOYSA-N 26 C13 13
1593 253.1301 257.1183 264.1669 268.1551 338926 369289 473371 305606 C11H16N4O3 1560 0.48 Histidinyl-Proline NC(CC1=CN=CN1)C(=O)N1CCCC1C(O)=O LNCFUHAPNTYMJB-UHFFFAOYSA-N 28.6 C13 9
180 308.0924 311.0835 318.126 321.1173 4825 7479 6747 6442 C10H17N3O6S 213 0.48 Glutathione N[[email protected]@H](CCC(=O)N[[email protected]@H](CS)C(=O)NCC(O)=O)C(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 11.8 C12 43
196 272.1731 277.1583 283.21 288.1953 2973 4999 4193 4694 C11H21N5O3 222 0.5 Arginyl-Proline NC(CCCNC(N)=N)C(=O)N1CCCC1C(O)=O LQJAALCCPOTJGB-UHFFFAOYSA-N 62.8 N15 28
180 152.0574 157.0425 157.0742 162.0595 2590 3359 3670 1638 C5H5N5O 143 0.5 Guanine NC1=NC(=O)C2=C(N1)N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0 C12 11
273 272.1731 277.1582 283.2099 288.195 9722 13926 13834 7080 C11H21N5O3 224 0.51 Prolyl-Arginine NC(=N)NCCCC(NC(=O)C1CCCN1)C(O)=O HMNSRTLZAJHSIK-UHFFFAOYSA-N 1.5 C12 43
180 175.1197 179.1079 181.1399 185.1281 12831 17475 18553 19686 C6H14N4O2 141 0.53 L-Arginine N[[email protected]@H](CCCN=C(N)N)C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-N 6.9 C12 131
1575 272.1724 277.1575 283.2092 288.1945 5083493 6237272 7491910 5875956 C11H21N5O3 1528 0.56 Arginyl-Proline NC(CCCNC(N)=N)C(=O)N1CCCC1C(O)=O LQJAALCCPOTJGB-UHFFFAOYSA-N 26.7 N15 157
3529 352.1666 355.1576 372.2338 375.2249 160772 145919 238006 177491 C20H21N3O3 3525 0.57 Phenylalanyl-Tryptophan NC(CC1=CC=CC=C1)C(=O)NC(CC1=CNC2=CC=CC=C12)C(O)=O JMCOUWKXLXDERB-UHFFFAOYSA-N 0 C13 23
259 308.0924 311.0836 318.126 321.1174 1730 2750 2586 32068 C10H17N3O6S 213 0.58 Glutathione N[[email protected]@H](CCC(=O)N[[email protected]@H](CS)C(=O)NCC(O)=O)C(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 11.8 C12 193
497 142.098 145.0891 148.1181 151.1093 43998 67416 66564 95564 C6H11N3O 517 0.6 L-Histidinol N[[email protected]](CO)CC1=CN=CN1 ZQISRDCJNBUVMM-YFKPBYRVSA-N 0 C12 37
8784 137.0461 141.0343 142.063 146.0512 23536 24881 37870 34265 C5H4N4O 8761 0.69 Allopurinol O=C1N=CN=C2NNC=C12 OFCNXPDARWKPPY-UHFFFAOYSA-N 0 C13 63
2290 302.1507 305.1419 318.2043 321.1956 488520 462842 787671 715698 C16H19N3O3 2319 0.69 Prolyl-Tryptophan OC(=O)C(CC1=CNC2=CC=CC=C12)NC(=O)C1CCCN1 UEKYKRQIAQHOOZ-UHFFFAOYSA-N 0 C12 59
273 269.1621 273.1502 281.2023 285.1904 7461 7597 12484 1362 C12H20N4O3 224 0.74 Leucyl-Histidine CC(C)CC(N)C(=O)NC(CC1=CN=CN1)C(O)=O XWOBNBRUDDUEEY-UHFFFAOYSA-N 0.9 N15 9
759 229.1553 231.1494 240.1923 242.1864 322383 247587 543330 339793 C11H20N2O3 804 0.75 Leucyl-Proline CC(C)CC(N)C(=O)N1CCCC1C(O)=O VTJUNIYRYIAIHF-UHFFFAOYSA-N 0 C12 88
250 288.2044 293.1895 300.2447 305.2299 5799 4910 10001 5679 C12H25N5O3 221 0.79 Arginyl-Leucine CC(C)CC(NC(=O)C(N)CCCNC(N)=N)C(O)=O WYBVBIHNJWOLCJ-UHFFFAOYSA-N 2.2 C12 34
7153 258.1107 259.1078 266.1376 267.1346 76771 94433 133143 105987 C8H20NO6P 7191 0.79 Glycerophosphocholine C[N+](C)(C)CCOP([O-])(=O)OC[[email protected]@H](O)CO SUHOQUVVVLNYQR-QMMMGPOBSA-N 0 C12 9
180 269.162 273.1502 281.2023 285.1906 1380 1911 2500 1267 C12H20N4O3 224 0.86 Leucyl-Histidine CC(C)CC(N)C(=O)NC(CC1=CN=CN1)C(O)=O XWOBNBRUDDUEEY-UHFFFAOYSA-N 0.9 N15 9
273 219.135 221.129 228.1652 230.1591 1090 876 1977 2337 C9H18N2O4 234 0.86 L-Lysopine CC(NC(CCCCN)C(O)=O)C(O)=O ZZYYVZYAZCMNPG-UHFFFAOYNA-N 6 C13 13
273 229.1556 231.1497 240.1926 242.1865 10214 7017 18710 6745 C11H20N2O3 227 0.87 Leucyl-Proline CC(C)CC(N)C(=O)N1CCCC1C(O)=O VTJUNIYRYIAIHF-UHFFFAOYSA-N 11.6 C13 41
497 319.1411 323.1293 334.1915 338.1796 106384 119051 199921 231679 C15H18N4O4 469 0.91 Tyrosyl-Histidine NC(CC1=CC=C(O)C=C1)C(=O)NC(CC1=CN=CN1)C(O)=O ZQOOYCZQENFIMC-UHFFFAOYSA-N 37.3 C13 74
3439 361.1993 367.1814 378.2563 384.2386 972695 952115 1880096 1180116 C17H24N6O3 3394 0.95 Arginyl-Tryptophan NC(CCCNC(N)=N)C(=O)NC(CC1=CNC2=CC=CC=C12)C(O)=O QADCERNTBWTXFV-UHFFFAOYSA-N 0 N15 126
230 229.1558 231.1499 240.1928 242.1869 8972 6588 17757 11917 C11H20N2O3 227 0.98 Leucyl-Proline CC(C)CC(N)C(=O)N1CCCC1C(O)=O VTJUNIYRYIAIHF-UHFFFAOYSA-N 11.6 C13 72
230 260.1617 263.153 271.1987 274.1898 1607 1416 3278 1953 C11H21N3O4 224 1.03 Hydroxyprolyl-Lysine NCCCCC(NC(=O)C1CC(O)CN1)C(O)=O BXAQOKHDAYJQPA-UHFFFAOYSA-N 2.4 C13 12
2503 253.119 255.1131 265.1593 267.1533 299685 423382 632684 516946 C12H16N2O4 2541 1.08 Tyrosyl-Alanine CC(NC(=O)C(N)CC1=CC=C(O)C=C1)C(O)=O NLKUJNGEGZDXGO-UHFFFAOYSA-N 0 C13 51
273 260.1616 263.1529 271.1986 274.1895 1358 1509 2876 459 C11H21N3O4 224 1.08 Hydroxyprolyl-Lysine NCCCCC(NC(=O)C1CC(O)CN1)C(O)=O BXAQOKHDAYJQPA-UHFFFAOYSA-N 2.4 C13 3
2211 361.1993 367.1815 378.2565 384.2386 210915 396508 453475 363396 C17H24N6O3 2201 1.1 Tryptophyl-Arginine NC(CC1=CNC2=C1C=CC=C2)C(=O)NC(CCCNC(N)=N)C(O)=O LCPVBXOHXMBLFW-UHFFFAOYSA-N 0 C13 30
1575 288.2035 293.1889 300.244 305.2292 1884540 2072277 4053500 2690484 C12H25N5O3 1564 1.1 Arginyl-Leucine CC(C)CC(NC(=O)C(N)CCCNC(N)=N)C(O)=O WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0 C12 73
497 229.1555 231.1495 240.1924 242.1865 115962 119944 254926 153113 C11H20N2O3 457 1.14 Leucyl-Proline CC(C)CC(N)C(=O)N1CCCC1C(O)=O VTJUNIYRYIAIHF-UHFFFAOYSA-N 10 N15 59
180 288.2042 293.1895 300.2446 305.2299 1300 1485 2904 2439 C12H25N5O3 221 1.16 Arginyl-Leucine CC(C)CC(NC(=O)C(N)CCCNC(N)=N)C(O)=O WYBVBIHNJWOLCJ-UHFFFAOYSA-N 2.2 C12 15
2308 195.1134 197.1074 205.1469 207.141 820378 984623 1877375 668150 C10H14N2O2 2335 1.19 Laccarin CC1CCNC2=C(C(C)=O)C(=O)NC12 DAMBAJDWLIFTNW-UHFFFAOYSA-N 0 C12 49
273 260.1981 263.1892 272.2384 275.2293 4328 5171 9932 2789 C12H25N3O3 224 1.2 Lysyl-Leucine CC(C)CC(NC(=O)C(N)CCCCN)C(O)=O ATIPDCIQTUXABX-UHFFFAOYSA-N 2.2 C13 17
230 322.189 327.174 337.2392 342.2244 3944 4831 9093 6691 C15H23N5O3 278 1.21 Arginyl-Phenylalanine NC(CCCNC(N)=N)C(=O)NC(CC1=CC=CC=C1)C(O)=O PQBHGSGQZSOLIR-UHFFFAOYSA-N 28.4 N15 40
2563 279.171 281.1652 294.2214 296.2155 288934 301991 703252 351849 C15H22N2O3 2534 1.28 Isoleucyl-Phenylalanine CCC(C)C(N)C(=O)NC(CC1=CC=CC=C1)C(O)=O WMDZARSFSMZOQO-UHFFFAOYSA-N 0 N15 31
558 260.1976 263.1887 272.2379 275.229 145455 179180 357184 179012 C12H25N3O3 587 1.3 Leucyl-Lysine CC(C)CC(N)C(=O)NC(CCCCN)C(O)=O OTXBNHIUIHNGAO-UHFFFAOYSA-N 0 C13 45
180 229.1557 231.1497 240.1927 242.1868 2654 2380 6625 2744 C11H20N2O3 227 1.32 Leucyl-Proline CC(C)CC(N)C(=O)N1CCCC1C(O)=O VTJUNIYRYIAIHF-UHFFFAOYSA-N 11.6 C13 18
3421 361.1993 367.1815 378.2563 384.2386 251942 381961 701564 1180116 C17H24N6O3 3394 1.48 Arginyl-Tryptophan NC(CCCNC(N)=N)C(=O)NC(CC1=CNC2=CC=CC=C12)C(O)=O QADCERNTBWTXFV-UHFFFAOYSA-N 0 N15 126
180 260.198 263.1893 272.2384 275.2296 988 1283 2824 652 C12H25N3O3 224 1.52 Lysyl-Leucine CC(C)CC(NC(=O)C(N)CCCCN)C(O)=O ATIPDCIQTUXABX-UHFFFAOYSA-N 2.2 C13 4

Table 1: Metabolite formulas and structures identified from LC-MS analysis of Bortezomib treated yeast using MISSILE and JUMPm.

Multiplexed quantification of Bortezomib regulated metabolites

We observed that higher Bortezomib concentrations had broader impacts on the yeast metabolic profile, with a greater proportion of metabolites showing a larger fold change in peak size relative to control (Figure 1i). The lowest concentration of drug had a relatively minor effect on metabolite levels, whereas the higher concentrations increasingly segregated metabolites into up- or down-regulated populations. This trend was also true for individual metabolites (Figure 1d). As expected we observed that Bortezomib treatment increased the levels of incomplete protein catabolites e.g., di-peptides (Figure 1j). Metabolites that were unaffected by drug-treatment included nucleo-bases (e.g. adenine) and other “housekeeping” metabolites such as glutathione. We also observed that membrane components and signaling lipids were strongly down-regulated with higher Bortezomib concentrations (Figure 1l and 1k; Table 1).

Advantages and limitations

The described multiplexed design has several advantages over classical unlabeled strategies for metabolite quantification. Peak alignment between analyses is a major confounding factor for unlabeled strategies, and much effort has been focused on resolving this issue. For multiplexed samples, peak alignment is unnecessary because the treatment conditions are analyzed simultaneously. Carbon and nitrogen stable isotope labeling does not affect the retention time of small molecules, so each labeled form co-elutes with the other treatment conditions. This co-elution is a major advantage because it controls for time and sample-dependent variations in retention time and ionization efficiency, a major source of technical error during metabolite quantification.

Using the current strategy we can expect to identify and quantify metabolites from four independent labeled conditions. The labels are comprised of light or heavy carbon and nitrogen atoms. Therefore we observe all four possible mass labels for a given metabolite if the chemical formula contains at least one nitrogen atom. We previously observed that ~50% of known metabolite formulas contain nitrogen while the rest do not. For metabolites without nitrogen, our strategy can only provide two independent labels. In that case, the two peaks represent the average of two cultures (i.e., cultures 1&2 or 3&4). Therefore the duplicate experimental design may be more suitable for studies focused on such metabolites.

We introduce a new capability to perform 4-plex relative quantification of metabolites in untargeted metabolomics experiments using the MISSILE strategy in combination with JUMPm software. Using this method, we can now analyze metabolites from two experimental conditions with duplicates, or with up to 4 independent experimental conditions (e.g. time series or dose response data). The isotope labels are used by JUMPm software to determine chemical formulas and perform relative quantification and false discovery control.

Acknowledgements

The authors thank Z. Wu for laboratory assistance, H. Tan, A. High and V. Pagala for MS instrument guidance, and other lab and facility members for helpful discussion. This work was partially supported by National Institutes of Health grants R01GM114260, R01AG047928, R01AG053987, R01AG047928, R01GM114260, the American Asthma Foundation (15-0020), and ALSAC (American Lebanese Syrian Associated Charities). The MS analysis was performed in the St. Jude Children’s Research Hospital Proteomics Facility, partially supported by NIH Cancer Center Support Grant (P30CA021765).

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