Kevin J. McHale1, Mark Szewc1, Gregory S. Stupp2, Chaevien Clendinen2, Ramadan Ajredini2, Arthur S. Edison2, Chris Beecher3
Appli cat i on N ote 5 9 2
Metabolomics of Hermaphroditic C. elegans via Isotopic Ratio Outlier Analysis (IROA) Using High-Resolution, Accurate-Mass LC-MS/MS
Thermo Fisher Scientific, Somerset, NJ; 2University of Florida, Gainesville, FL; 3IROA Technologies, Ann Arbor, MI
1
Key Words Isotopic Ratio Outlier Analysis (IROA ), Untargeted metabolomics, Caenorhabditis elegans, High-Resolution, Accurate-Mass (HR/AM) liquid chromatography-tandem mass spectrometry, Q Exactive, Ascarosides, ClusterFinder™ ®
Goal The goal of this study was to investigate metabolic changes in hermaphroditic C. elegans in an untargeted manner using isotopic labeling and high-resolution, accurate-mass (HR/AM) mass spectrometry.
Introduction Caenorhabditis elegans is one of the best-studied animals in science and the genetics of this “worm” is especially well defined.1 Despite this, metabolomic studies in C. elegans have only recently become an active area of research. The Isotopic Ratio Outlier Analysis (IROA®) protocol uses highly patterned 13C isotopic signatures to identify and quantitate metabolites (Figure 1).2
Based on the IROA pattern seen in mass spectral analysis, both the accurate mass and the number of carbons in any molecule are determined. The combined information can significantly improve the accuracy of formula determination. The inclusion of a biologically similar internal standard, i.e. the control IROA 95% 13C-labeled sample, increases the accuracy of quantitation by making every measurement relative to an appropriate isotopic standard. The use of an isotopic IROA standard also reduces error introduced during sample preparation and analysis including ionization suppression. The marriage of IROA and HR/AM liquid chromatography-tandem mass spectrometry (LC-MS/MS) with C. elegans metabolomics allows experiments that assess the biological response to stresses or stimuli. When done conventionally, these experiments would be difficult due to interferences by metabolites of unlabeled organisms. With IROA labeling and HR/AM detection, metabolites can be distinguished in an untargeted manner, quantitated, and identified to their chemical formulas.
Figure 1. IROA experimental design. The IROA protocol uses 13C-isotopic media to create IROA signatures in all metabolites by allowing the bacterial diet of the worms to grow in either a 5% or a 95% 13C enriched media. Once labeled, the worms and their metabolites are also fully labeled. The 5% labeled worm metabolites contribute the lower mass (left-hand) portion of the IROA pattern. The 95% labeled metabolites contribute the higher mass (right-hand) side of the IROA pattern. If the 95% is the control and thus constant, the 5% experimental is always measured against a common denominator.
2
Experimental
Table 2. Scan parameters for Q Exactive mass spectrometer
Sample Preparation E. coli grown in minimal media supplemented with 95% or 5% randomly 13C-labeled glucose (IROA media) was fed to wild-type C. elegans hermaphrodites. After worms reached young adult stage, the 5%-labeled worm population was split into four replicates and then incubated with male worms. The 95%-labeled worms served as a control and were not challenged. The two worm populations were mixed in a 1:1 ratio and harvested. The recovered supernatant and worm pellets from the biological replicates were dried under nitrogen stream and stored at –80 °C. Prior to LC-MS/MS analyses, samples were thawed and reconstituted with 28 µL 50% acetonitrile/50% water. Liquid Chromatography Samples were separated by injection of 2 µL via reversephase LC (RPLC) and hydrophilic interaction liquid chromatography (HILIC) using the Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC system. RPLC was performed on a Thermo Scientific™ Hypersil™ GOLD aQ 2.1 mm × 150 mm, 1.9 µm column with a gradient of 0–95% B (A: 0.1% formic acid; B: acetonitrile + 0.1% formic acid) in 12 min. HILIC was performed on a proprietary 2.1 × 150 mm column using a gradient of 10–70% A over 15 min, where A was 10 mM ammonium acetate adjusted to basic pH (B: acetonitrile).
Parameter Setting Full-scan MS Mass range:
HILIC
RPLC
m/z 70–1,000 m/z 70–1,000
Resolution (FWHM at m/z 200):
70,000 70,000
AGC target:
1 x 106
1 x 106
50
50
Max inject time (ms):
Data-dependent MS2
HILIC RPLC
Resolution (FWHM at m/z 200):
17,500 17,500
AGC target:
1 x 105
1 x 105
150
150
4
2
1.5
1.5
Max inject time (ms): Top N MS/MS: Isolation width (Da): Collision energy (NCE):
35
35
Stepped NCE (%):
40
40
Underfill (%):
25
50
Dynamic Exclusion duration (s):
6
4
1–6
0.2–2
Apex trigger (s):
Data Analysis The data were analyzed using IROA ClusterFinder™ software. The software detects and characterizes IROA Mass Spectrometry peaks, which are visible in the mass spectra due to the All data were acquired on the Thermo Scientific™ enriched levels of 13C. As the IROA peaks are all Q Exactive™ mass spectrometer using external mass mathematically calculable, the IROA ClusterFinder calibration. Sample analyses were conducted in both software algorithms achieved a data reduction of complex positive and negative electrospray ionization (ESI) modes raw data to concise, high-value information by as separate LC-MS/MS runs. Full-scan MS were collected characterizing all peaks according to source (artifact, at a mass resolution of 70,000 FWHM at m/z 200. experimental (12C), control (13C), or standard); removing Data-dependent MS/MS scans were acquired at all artifacts; aligning and pairing all remaining peaks 17,500 FWHM. Relevant source and scan parameters across all scans, identifying and determining the relative are shown in Tables 1 and 2. 12 C/13C ratios of analytes in each sample. The number of Table 1. Source parameters for Heated Electrospray Ionization carbons for each biochemical compound was calculated probe (HESI-II) from its mass spectra by the distance between the two monoisotopic peaks, 12C and 13C. IROA components were Parameter Setting searched against the KEGG™ database to determine HILIC RPLC compound identities based on the elemental compositions Sheath gas: 45 60 as established by HR/AM. Aux gas: 20 20 Sweep gas
2
2
Vaporizer temperature (°C):
350
450
Capillary temperature (°C):
275
275
S-Lens (%):
40
40
+3000 (-2250)
+3000 (-2250)
Spray voltage (V)
Results and Discussion
3
C and 13C peaks. This indicates that many of the statistically relevant compounds in these experiments have yet to be positively identified. Given that many of these unknown compounds may be secondary metabolites and potentially novel, this is not surprising. Interpretation of the HR/AM spectra and MS/MS data are on-going. 12
Table 3. IROA components observed in 13C-labeled hermaphroditic C. elegans via HILIC and RPLC
Number of IROA Components Observed
HILIC
RPLC
3 of 4 samples
4021
5090
Named
668
674
2-fold change
1303
1409
2-fold change, named
81
100
Unique to LC method
20
39
The number of IROA peaks identified in three of four samples through either HILIC or RPLC represented a significant reduction from the millions of mass spectral peaks in an average chromatographic run. While the number of observed IROA components in RPLC is about 25% higher than with HILIC, the total number of named components, as determined with a KEGG database search, was comparable. A statistical t-test evaluation was conducted to ascertain the IROA components that showed at least a 2-fold change (p-value ≤ 0.05) in the challenged hermaphroditic C. elegans samples. The RPLC method again shows a slight advantage in the number of IROA components displaying a 2-fold change. Inspection of the named IROA components with a statistical change reveals two interesting findings. First, many of the components unique to RPLC are lipophilic in nature (for example, fatty acids and ascarosides). The fact that a greater number of named and unique components were observed by RPLC suggests that these lipophilic compounds play a significant role in the interactions between hermaphroditic and male nematodes. Indeed, the ascarosides are a known class of signaling compounds that regulate behavior in C. elegans.3 Second, less than 10% of the IROA components showing a 2-fold change were identified in the KEGG database, but for all of these the most likely formula was determined given the dual constraints of the accurate mass and carbon number derived from the distance between the monoisotopic
RT: 0.00 - 4.00 RT: 0.75 100 Relative Abundance
HILIC versus RPLC Owing to the widely varying polarities of the compounds in metabolomics experiments, complementary reversephase LC and HILIC methods were employed to measure the IROA-labeled samples. Table 3 shows a summary of the results.
Figure 2 displays the extracted ion chromatograms (XICs) for carnitine and 2-aminoadipic acid by RPLC and HILIC, respectively. These two compounds were determined to be statistically changing by IROA measurements. There are several interesting LC/MS experimental findings regarding these two components. First, these isobaric compounds coelute with RPLC (near the void volume). Nevertheless, the ultra-high resolution of the Q Exactive mass spectrometer easily mass resolves these two isobaric ions and all their 13C isotopes from the IROA labeling at 70,000 FWHM (Figure 3). Second, these components are chromatographically separated by HILIC. The identities of the major HILIC peaks were confirmed by HR/AM MS/MS with library matching (data not shown). Third, close inspection of the HILIC data show at least two other isomeric species of 2-aminoadipic acid. Data-dependent HR/AM MS/MS of m/z 162.0760 at 4.92 min and 5.32 min are presented in Figures 4 and 5, respectively. Neither the Thermo Scientific Metabolomics MS/MS library nor the online METLIN MS/MS library had a match for these fragment ion spectra. Manual interpretation has putatively identified them as O-acetylhomoserine and glutamate methyl ester.
m/z 162.1125
80 60
NL: 4.63E8 m/z= 162.1117-162.1133 F: FTMS + p ESI Full ms [70.00-1000.00] MS ICIS RPLC_PosESI_95N2_5H2 _P_2005
RT: 0.00 - 10.00
60 40 20
m/z 162.0761
80 60
NL: 6.38E8 m/z= 162.0753-162.0769 F: FTMS + p ESI Full ms [70.00-1000.00] MS ICIS RPLC_PosESI_95N2_5H2 _P_2005
40
0 100
RT: 7.42
m/z 162.0761
80 60
NL: 8.98E8 m/z= 162.1117-162.1133 F: FTMS + p ESI Full ms [70.00-1000.00] MS ICIS HILIC_PosESI_95N2_5H2 _P_2003
NL: 5.17E8 m/z= 162.0753-162.0769 F: FTMS + p ESI Full ms [70.00-1000.00] MS ICIS HILIC_PosESI_95N2_5H2 _P_2003
40
20 0
m/z 162.1125
80
20 RT: 0.74
RT: 6.23
100
40
0 100 Relative Abundance
Removal of Artifacts, Noise, and Sample-to-Sample Analytical Variance The IROA protocol utilized an enrichment of 95% and 5% 13C for the control and experimental populations. This created readily recognizable isotopic patterns (Figure 1) that were used to 1) discriminate control from experimental samples and thereby allow composite pooling to remove sample-to-sample analytical variance and the effects of ion suppression, and 2) distinguish noise and artifactual peaks from IROA-labeled biological peaks. The later ensured a significant reduction of the number of peaks in the dataset as only biological peaks were interrogated.
20 RT: 0.89 RT: 1.94 0
1
2 Time (min)
3
4
0
RT: 5.32 RT: 4.92 0
5 Time (min)
10
Figure 2. XICs for carnitine and 2-aminoadipic acid by RPLC (left) and HILIC (right). Chromatograms are ±5 ppm.
RPLC_PosESI_95N2_5H2_P_2005 #264 RT: 0.75 AV: 1 NL: 5.69E8 T: FTMS + p ESI Full ms [70.00-1000.00] 162.0760 C 6 H 12 O 4 N -0.7781 ppm 100 90
2-Aminoadipic acid
Relative Abundance
80 70
4
162.1122 C 7 H 16 O 3 N -1.4896 ppm
Carnitine
60 50 40 30 20 10 0 161.90
161.95
162.00
162.05
162.10 m/z
162.15
162.20
RPLC_PosESI_95N2_5H2_P_2005 #264 RT: 0.75 AV: 1 NL: 5.69E8 T: FTMS + p ESI Full ms [70.00-1000.00] 2-Aminoadipic 162.0760 100 acid
162.25
Carnitine
Relative Abundance
80 60 40 20
163.1156 164.1192
0 161
162
163
12C-
164
165
side
m/z
166
169.1358
168.0961
167.1291
165.1224
167
168
169
13C-
170
side
Figure 3. HR/AM of carnitine and 2-aminoadipic acid at 70,000 FWHM by RPLC. The top spectrum is a close-up of monoisotopic masses; the bottom spectrum shows the full IROA envelope.
116.0707
HILIC_PosESI_95N2_5H2_P_2003 #1923 RT: 4.92 AV: 1 NL: 2.20E6 T: FTMS + p ESI d Full ms2
[email protected] [50.00-185.00] 84.0444 C4 H6 O N 0.1511 ppm 100
84.0444
90
Relative Abundance
80 70
102.0550
60 50
102.0550 C4 H8 O2 N 0.8921 ppm
40 30 20 10 0
144.0655 C 6 H 10 O 3 N -0.2011 ppm 116.0707 C 5 H 10 O 2 N 0.5685 ppm
56.0498 C3 H6 N 4.9153 ppm 50
60
70
80
90
100
110
120 m/z
162.0759 C 6 H 12 O 4 N -1.4371 ppm 130
140
150
160
170
180
Figure 4. HR/AM MS/MS of 2-aminoadipic acid isomer at 4.92 min by HILIC. Spectrum is consistent with O-acetylhomoserine.
130.0499
HILIC_PosESI_95N2_5H2_P_2003 #2130 RT: 5.37 AV: 1 NL: 8.16E6 T: FTMS + p ESI d Full ms2
[email protected] [50.00-185.00] 84.0444 C4 H6 O N 0.2418 ppm 100 90
116.0707 C 5 H 10 O 2 N 1.0943 ppm
Relative Abundance
80 70 60 40 20 10 0
116.0707
130.0499 C5 H8 O3 N -0.0301 ppm
50 30
84.0444
145.0496 C6 H9 O4 0.4899 ppm
56.0498 C3 H6 N 5.0514 ppm 50
60
70
80
90
100
110
120 m/z
130
140
150
162.0760 C 6 H 12 O 4 N -0.7781 ppm
160
170
180
Figure 5. HR/AM MS/MS of 2-aminoadipic acid isomer at 5.32 min by HILIC. Spectrum is consistent with glutamate methyl ester.
Ascarosides In recent years, ascarosides have been identified as signaling pheromones in C. elegans.3 These pheromones are responsible for various social interactions, including sexual attraction and repulsion in hermaphroditic nematodes. Figure 6 shows the RPLC chromatograms for several ascarosides that are up-regulated in the pellet of the 5% 13C-labeled hermaphrodite samples. Note that RPLC is the preferred method for the ascarosides, as HILIC does not separate structural isomers observed via RPLC (data not shown). One of the ascarosides, icas#9, is an indole ascaroside, whose structure was confirmed by HR/AM MS/MS (Figure 7). The icas#9 ascaroside has been shown to promote attraction and aggregation in hermaphroditic C. elegans.4 The same research group also showed that ascr#3, a strongly repulsive ascaroside, serves to balance the attractive effect of icas#3 at high concentrations. The presence of increased concentrations of ascr#9 in these samples may serve a similar purpose, but this supposition warrants further investigation. RT: 0.00 - 10.00 100
m/z 233.1031 (-)
Relative Abundance
0 100 0 100
oscr#11
ascr#11
RT: 3.73 RT: 3.90
ascr#9
m/z 273.1344 (-)
0 100
m/z 275.1500 (-)
0 100
m/z 390.1558 (-)
oscr#9
RT: 4.64
m/z 269.1360 (+)
0 100
0
RT: 3.04
m/z 247.1187 (-)
0 100
ascr#2 RT: 5.58
7.95
ascr#7 RT: 5.94
ascr#1 RT: 8.05
icas#9 RT: 8.32
m/z 331.2126 (-) 0
1
ascr#18 2
3
4
5 Time (min)
6
7
oscr#18
8
9
10
Figure 6. XICs of ascarosides, RPLC. Data from C. elegans pellet. Chromatograms are ±5 ppm.
RPLC_NegESI_95N2_5H2_P_2006 #2703 RT: 8.06 AV: 1 NL: 1.32E6 T: FTMS - p ESI d Full ms2
[email protected] [50.00-415.00] 247.1187 C 11 H 19 O 6 73.0296 -0.1779 ppm 100 C 3 H 5 O 2 1.2553 ppm 160.0405 90 C9 H6 O2 N 80 0.4032 ppm Relative Abundance
5
70
50
73.0296
160.0405 -CO 2
116.0507 C8 H6 N 0.8777 ppm
60
247.1187
390.1566 C 20 H 24 O 7 N 2.1053 ppm
116.0507
40 30 290.1043 C 15 H 16 O 5 N 3.0426 ppm
20 10 0
50
100
150
200
m/z
250
300
350
400
Figure 7. HR/AM MS/MS, icas#9. m/z 73.0296 is a diagnostic negative ion fragment for all ascarosides.
References
The IROA protocol enabled accurate metabolite measurement in the study through the following:
1. Brenner, S. The Genetics of Caenorhabditis elegans. Genetics 1974, 77(1), 71–94.
• Pooling control and experimental samples prior to sample extraction and LC-MS/MS analysis and reducing sample-to-sample variance and the impact of ion suppression
2. de Jong, F.A. and Beecher, C. Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis (IROA®), an isotopic-labeling technique for accurate biochemical profiling. Bioanalysis 2012, 4(18), 2303–14. PMID: 23046270
• Discriminating biosynthesized molecules and artifacts and noise • Determining the number of carbons in each biological molecule, constraining the possible number of formulae • Providing relative quantitation by determining the 12 C/13C ratios of analytes in each sample IROA labeling and the ClusterFinder software together with HR/AM on the Q Exactive mass spectrometer identified >1300 components that are statistically changing by at least 2-fold in hermaphroditic C. elegans. RPLC and HILIC are complementary LC methods that are necessary to provide a complete metabolomics picture for hydrophilic and lipophilic compounds. The HR/AM MS/MS data provided confirmations for IROA component identifications, and structural information for unknown compound identifications.
3. Ludewig, A.H. and Schroeder F.C. Ascaroside signaling in C. elegans (Jan. 18, 2013), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.155.1, http://www. wormbook.org 4. Srinivasan, J.; von Reuss, S.H.; Bose, N.; Zaslaver, A.; Mahanti, P.; Ho, M.C.; O’Doherty, O.G.; Edison, A.S.; Sternberg, P.W.; Schroeder, F.C. A Modular Library of Small Molecule Signals Regulates Social Behaviors in Caenorhabditis elegans. PLoS Biol. 2012, 10(1), doi: 10.1371/journal.pbio.1001237.
Ascarosides were a key class of signaling compounds observed to be statistically changing in hermaphroditic C. elegans. Future work will examine the specific and synergistic nature of target ascarosides on C. elegans’ behavior.
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Appli cat i on N ote 5 9 2
Conclusion
