Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass SpectrometryJunhua Wang,1 Terri Christison,1 Kaori Misuno,2 Shen Hu,2 Ralf Tautenhahn,1 Linda Lopez,1 Yingying Huang1 1Thermo Fisher Scientific, San Jose, CA; 2School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA

2 Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry

Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry Junhua Wang1, Terri Christison1, Kaori Misuno2, Shen Hu2, Ralf Tautenhahn1, Linda Lopez1, Yingying Huang1 1Thermo Fisher Scientific, San Jose, CA; 2School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA

Conclusion • An untargeted metabolomic analysis of head and neck cancer cells has been

performed in this study.

• The outstanding resolution of IC has led to the differentiation of many isobaric and isomeric polar metabolites.

• Large-scale validation and in-depth integration of the metabolomics data with proteomics and transcriptomic data is to be conducted.

References 1. Lorenz, M.A.et al Anal. Chem., 2011, 83, 3406–3414

2. Tautenhahn, R. et al Anal. Chem., 2011, 83, 696-700

This work is published in Anal. Chem. DOI: 10.1021/ac500951v

Overview Purpose: Demonstrate Cap IC coupling with high-resolution, accurate-mass (HRAM) MS for untargeted metabolomics research analysis and the unique capability to differentiate isobaric metabolites.

Methods: A Thermo Scientific™ Dionex™ ICS-4000 capillary IC system coupled with a Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer for cell lysate detection and differential analysis.

Results: Metabolites were detected at low concentration 60 ppt (0.2 to 3.4 fmol on column) with S/N varying from 3-20. The LODs were in the range of 0.04 to 0.5 nmol/L. Outstanding separation for anionic polar metabolites and superior analytical sensitivities were achieved compared to reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC).

Introduction The metabolomics approach has obtained increasing attention in oral cancer study. A highly analytically sensitive research platform coupling capillary ion chromatography (Cap IC) with the Q Exactive mass spectrometer has been developed, which is applied to potential metabolic biomarkers profiling of oral squamous cell carcinoma (OSCC) metastasis from cell lysates. Cap IC has demonstrated outstanding separation for anionic polar metabolites and superior analytical sensitivities compared to RPLC and HILIC methods. Differential analysis finds significant changes in energy metabolism pathways, e.g., glycolysis cycle and tricarboxylic acid cycle. The experiments demonstrate Cap IC as a complementary separation technique by providing superior resolution and analytical sensitivity of polar metabolites combined with the high-resolution, accurate-mass measurement capabilities to differentiate isobaric metabolites.

Methods Sample Preparation

Three OSCC cell lines, UMSCC1, UMSCC5, cancer stem-like cells (CSC), and according wild-type controls with biological replicates were harvested and counted. Cellular metabolites were extracted using a liquid nitrogen snap-freezing method with methanol/water according the literature.1

Ion Chromatography and Liquid Chromatography

An ICS-4000 system with an electrolytic suppressor was used to convert the potassium hydroxide gradient to pure water. The Cap IC analysis was performed on a Thermo Scientific™ Dionex™ IonPac™ AS11HC-4µm, 0.4 x 250 mm column. The IC flow rate was 25 µL/min, supplemented post-column with 10 µL/min make-up flow of methanol/acetic acid (2 mM). The gradient started with 2 mM potassium hydroxide, rose to 12 mM at 13.5 min, then to 20 mM at 22.5 min and to 70 mM at 31.5 min. It was then held at 70 mM for 6 min, followed by a decrease to 2 mM in 0.1 min and held for 7.5 min to re-equilibrate the column. HILIC was performed on a 150 x 2.1 mm, 5 µm SeQuant®- ZIC®-pHILIC column at 250 µL/min. UHPLC on a Thermo Scientific™ Hypersil GOLD™ 150 x 2.1 mm, 1.9 µm column at 450 µL/min. LC was performed on a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC HPG system.

Mass Spectrometry

A Q Exactive quadrupole-Orbitrap mass spectrometer was operated under ESI negative mode for all detections. Full mass scan (m/z 67-1000) used resolving power 70,000 (at m/z 200) with automatic gain control (AGC) target of 1 x 106 ions and a maximum ion injection time (IT) of 50 ms. Data-dependent MS/MS was acquired on a “Top10” data-dependent mode using the following parameters: resolving power, 17,500; AGC, 1 x 105 ions; maximum IT, 100 ms; isolation window,1.5 amu; normalized collision energy, 35% ± 50%; underfill ratio, 1.0%. Source ionization parameters were optimized according to different flow rates. For all capillary flow IC, the settings were as follows: spray voltage, -2.8 kV; transfer capillary temperature, 300 ºC; S-lens level, 50; heater temperature, 125 ºC; sheath gas, 26; aux gas, 2. For higher flow LC, the following settings were used: spray voltage, -3.2 kV, heater temperature, 275 ºC; sheath gas,45; aux gas, 5.

Results Cap IC/MS Analysis of Metabolite Standards

The Cap IC – Q Exactive platform showed an outstanding separation and detection analytical sensitivity for polar metabolites. Metabolites were detected at 60 ppt (0.2 to 3.4 fmol on column), with S/N varying from 3-20. The LODs were in the range of 0.05 to 0.5 nmol/L. Isomeric compounds including: (a) α-D-glucose-1-phosphate, α-D-glucose-6-phosphate, and D-fructose-6-phosphate; (b) citrate and isocitrate; (c) trans- and cis-aconitate; and (d) fructose-1,6-phosphate and fructose-2,6-phosphate were baseline resolved. The resolution did not worsen even when the concentrations were lowered 10,000-fold, and the RT shifts were less than 0.04 min. HILIC using a ZIC-pHILIC column were performed afterwards on the same Q Exactive MS. UHPLC separation for the polar compounds was mostly unacceptable (data not shown), while HILIC showed good separation for most metabolites. However, the resolution on certain isomers like sugar phosphates and cis- and trans-aconitate were much worse compared to Cap IC. The analytical sensitivity by Cap IC system is generally 10-100 times better than the HILIC method.

FIGURE 1. Schematic diagram of capillary IC – Q Exactive platform for untargeted metabolic profiling

Data Analysis

Differential analyses were performed using Thermo Scientific™ SIEVE™ software version 2.1. Statistical results, putative IDs, and pathways were generated by searching ChemSpider® and KEGG™ databases. Metabolites of interest were identified via MS/MS mass spectral database matching. The raw data were converted to mzXML format using ProteoWizard and analyzed by XCMS Online and metaXCMS [2].

Peak # Metabolite Name Formula M-H On column (fmol) LOD

(S/N = 3,

nM)

1 D-Glucose C6H12O6 179.0561 0.17 0.3

2 Mevalonate C6H12O4 147.0663 2.0 0.1

3 Lactate C3H6O3 89.0244 3.4 0.1

4 Uridine C9H12N2O6 243.0623 1.2 0.25

5 α-D-Glucose 1-phosphate C6H13O9P 259.0224 1.2 0.2

6 α-D-glucose-6-phosphate C6H13O9P 259.0224 1.2 0.2

7 D-Fructose 6-phosphate C6H13O9P 259.0224 1.2 0.2

8 Adenosine 3'-5'-cyclic monophosphate (cAMP) C10H12N5O6P 328.0452 0.91 0.2

9 Tartrate C4H6O6 149.0092 2.0 0.5

10 2-oxoglutarate C5H6O5 145.0142 2.1 0.2

11 Adenosine 5'-monophosphate (AMP) C10H14N5O7P 346.0558 0.87 0.1

12 2-phosphoglycerate C3H7O7P 184.9857 1.6 0.3

13 Citrate C6H8O7 191.0197 1.6 0.2

14 Isocitrate C6H8O7 191.0197 1.6 0.05

15 cis-Aconitate C6H6O6 173.0092 1.7 0.2

16 trans-Aconitate C6H6O6 173.0092 1.7 0.2

17 Phosphoenolpyruvate C3H5O6P 166.9751 1.8 0.2

18 D-Fructose-1,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

19 D-Fructose-2,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

20 Dihydroxy acetone-Phosphate C3H7O6P 168.9908 1.8 0.04

21 Inosine 5'-monophosphate C10H13N4O8P 347.0398 0.87 0.1

FIGURE 2.Separation of 21 standard metabolites at 600 ppb by Cap IC (A), 60 ppt by Cap IC (B) and 600 ppb by HILIC method (C) with Orbitrap MS Injection volume 5 µL. Standard mixture for Cap IC was diluted in water; for HILIC, the mixture was diluted in acetonitrile/water (3:1, v/v).

FIGURE 3. Inter-day reproducibility analysis of the metabolite standards at 600 ppb by Cap IC with conductive detection (n=6). RSDs of intensity were 5.5%, 7.8%, and 6.0% and RSDs of RT were 6.5%, 8%, and 7.2 %, respectively, for three inorganic ions chloride, carbonate, and phosphate coexisting in the sample solution.

FIGURE 4. (A) The overlap of detected metabolite number in cell lysate by Cap IC and LC methods. (B) The overlap of aligned features among three pairs of cancer cell samples by metaXCMS analysis.

FIGURE 5. Separation of peaks corresponding to m/z 259.0224 in UM1 oral cancer cells by (A) Cap IC, (B) UHPLC, (C) Cap-LC, (D) ZIC-pHILIC, and (E) Cap-HILIC. The five methods were connected to the same Q Exactive MS.

Metabolic Profiling of Cell Lysates by Cap IC

We used 42 standards including isomeric compounds (corresponding to 36 m/z values) to evaluate the system performance. The same m/z list was used to extract the peaks from the UM1 cell sample from Cap IC, UHPLC, and HILIC analyses. As shown in Figure 4A, for this list of polar compounds Cap IC detected 65 peaks, ZIC-pHILIC detected 38 peaks, and UHPLC detected 29 peaks. A total of 26 peaks were detected by all three methods, but Cap IC showed much greater coverage. Cap IC detected 25 more peaks than HILIC with only one compound, acetyl-CoA, detected uniquely by HILIC. This is likely because the acetyl-CoA molecules are too large for this particular IC column to retain.

Differential Analysis, Pathway Mapping and Meta-Analysis

MetaXCMS software for second-order analysis of global metabolomic data was used to perform the meta-analysis.2 All three datasets were individually analyzed with XCMS Online, which had detected 11377, 13302, and 10532 aligned features for UM1, UM5, and NCSC sample groups, respectively. The results were combined and analyzed by metaXCMS. Figure 4B shows the significant features (P-value < 0.05, ratio > 2) in each sample set, and a total of 218 features were identified as the common ones for all three sample groups.

FIGURE 6. Chromatogram overlays of the 11 sugar monophosphates in three pairs of cancer cell samples. (A) Cancer stem cells (CSCs) versus non-stem cancer cells (NSCCs); (B) UM1 versus UM1-KD; (C) UM5 versus UM5-KD. The inserts in each figure are the peak overlays of the internal standard hippuric acid-d5 ([M-H]-183.0824) that was spiked in the cell lysate samples, which showed a very reproducible intensity.

Peak # Obs. [M-H]- Δ ppm Compound Identity Matching UM1/KD UM5/KD CSC / NSCC

RT MS/MS ratio P-value ratio p-value ratio P-value

1 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 0.49 0.002 0.38 <0.0001 0.98 0.9

2 259.0225 0.4 Unknown n/a + 1.7 0.07 1.8 0.46 1.4 0.003

3***

259.0225 0.5 α-D-Glucose 1-phosphate + +

2.5 0.01 1.8 0.008 4.5 <0.000

1

4 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 1.5 0.02 0.56 0.04 1.0 0.5

5

259.0226 0.7 D-Mannose 1-phosphate n/a +

2.4 <0.0001 1.6 <0.0001 0.67 <0.000

1

6 259.0225 0.5 D-myo-Inositol-4-phosphate n/a + 2.0 0.003 1.6 0.002 3.0 0.4

7

259.0226 0.8 α-D-Fructose 1-phosphate + +

1.1 0.3 0.56 0.0002 0.63 <0.000

1

8 259.0226 0.9 α-D-Galatose 1-phosphate + + 0.95 0.6 0.15 <0.0001 - -

9*** 259.0226 0.8 D-Fructose 6-phosphate + + 3.4 0.003 3.9 0.0001 16 0.0002

10*** 259.0226 0.8 α-D-Glucose 6-phosphate + + 3.2 0.001 3.3 0.009 11 0.0006

11*** 259.0226 0.9 D-Mannose 6-phosphate n/a + 1.8 0.07 2.1 0.004 4.0 0.0006

FIGURE 7. Cap IC/MS analysis revealed metabolic changes of the glycolysis pathway between oral CSCs and NSCCs. p: p-value, R: fold change

Table 2. Differential changes of 11 sugar monophosphates between three pairs of cellular samples. RT: retention time of standards; n/a: RT not available; *** represents those peaks sharing the same trends in multiple cell lines.

For Research use only. Not for use in diagnostic procedures. SeQuant and ZIC are registered trademarks of Merck Millipore International. Chempsider is a registered trademark of ChemZoo Inc. KEGG is a trademark of Kyoto University, Kanehisa Lab. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64131-EN 0614S

TABLE 1. Twenty-one metabolite standards used in Figure 2 and their LOD by Cap IC-Q Exactive MS

(A) (B) (C) (A) (B) (C)

3Thermo Scientific Poster Note • PN-64131-ASMS-EN-0614S

Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry Junhua Wang1, Terri Christison1, Kaori Misuno2, Shen Hu2, Ralf Tautenhahn1, Linda Lopez1, Yingying Huang1 1Thermo Fisher Scientific, San Jose, CA; 2School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA

Conclusion • An untargeted metabolomic analysis of head and neck cancer cells has been

performed in this study.

• The outstanding resolution of IC has led to the differentiation of many isobaric and isomeric polar metabolites.

• Large-scale validation and in-depth integration of the metabolomics data with proteomics and transcriptomic data is to be conducted.

References 1. Lorenz, M.A.et al Anal. Chem., 2011, 83, 3406–3414

2. Tautenhahn, R. et al Anal. Chem., 2011, 83, 696-700

This work is published in Anal. Chem. DOI: 10.1021/ac500951v

Overview Purpose: Demonstrate Cap IC coupling with high-resolution, accurate-mass (HRAM) MS for untargeted metabolomics research analysis and the unique capability to differentiate isobaric metabolites.

Methods: A Thermo Scientific™ Dionex™ ICS-4000 capillary IC system coupled with a Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer for cell lysate detection and differential analysis.

Results: Metabolites were detected at low concentration 60 ppt (0.2 to 3.4 fmol on column) with S/N varying from 3-20. The LODs were in the range of 0.04 to 0.5 nmol/L. Outstanding separation for anionic polar metabolites and superior analytical sensitivities were achieved compared to reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC).

Introduction The metabolomics approach has obtained increasing attention in oral cancer study. A highly analytically sensitive research platform coupling capillary ion chromatography (Cap IC) with the Q Exactive mass spectrometer has been developed, which is applied to potential metabolic biomarkers profiling of oral squamous cell carcinoma (OSCC) metastasis from cell lysates. Cap IC has demonstrated outstanding separation for anionic polar metabolites and superior analytical sensitivities compared to RPLC and HILIC methods. Differential analysis finds significant changes in energy metabolism pathways, e.g., glycolysis cycle and tricarboxylic acid cycle. The experiments demonstrate Cap IC as a complementary separation technique by providing superior resolution and analytical sensitivity of polar metabolites combined with the high-resolution, accurate-mass measurement capabilities to differentiate isobaric metabolites.

Methods Sample Preparation

Three OSCC cell lines, UMSCC1, UMSCC5, cancer stem-like cells (CSC), and according wild-type controls with biological replicates were harvested and counted. Cellular metabolites were extracted using a liquid nitrogen snap-freezing method with methanol/water according the literature.1

Ion Chromatography and Liquid Chromatography

An ICS-4000 system with an electrolytic suppressor was used to convert the potassium hydroxide gradient to pure water. The Cap IC analysis was performed on a Thermo Scientific™ Dionex™ IonPac™ AS11HC-4µm, 0.4 x 250 mm column. The IC flow rate was 25 µL/min, supplemented post-column with 10 µL/min make-up flow of methanol/acetic acid (2 mM). The gradient started with 2 mM potassium hydroxide, rose to 12 mM at 13.5 min, then to 20 mM at 22.5 min and to 70 mM at 31.5 min. It was then held at 70 mM for 6 min, followed by a decrease to 2 mM in 0.1 min and held for 7.5 min to re-equilibrate the column. HILIC was performed on a 150 x 2.1 mm, 5 µm SeQuant®- ZIC®-pHILIC column at 250 µL/min. UHPLC on a Thermo Scientific™ Hypersil GOLD™ 150 x 2.1 mm, 1.9 µm column at 450 µL/min. LC was performed on a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC HPG system.

Mass Spectrometry

A Q Exactive quadrupole-Orbitrap mass spectrometer was operated under ESI negative mode for all detections. Full mass scan (m/z 67-1000) used resolving power 70,000 (at m/z 200) with automatic gain control (AGC) target of 1 x 106 ions and a maximum ion injection time (IT) of 50 ms. Data-dependent MS/MS was acquired on a “Top10” data-dependent mode using the following parameters: resolving power, 17,500; AGC, 1 x 105 ions; maximum IT, 100 ms; isolation window,1.5 amu; normalized collision energy, 35% ± 50%; underfill ratio, 1.0%. Source ionization parameters were optimized according to different flow rates. For all capillary flow IC, the settings were as follows: spray voltage, -2.8 kV; transfer capillary temperature, 300 ºC; S-lens level, 50; heater temperature, 125 ºC; sheath gas, 26; aux gas, 2. For higher flow LC, the following settings were used: spray voltage, -3.2 kV, heater temperature, 275 ºC; sheath gas,45; aux gas, 5.

Results Cap IC/MS Analysis of Metabolite Standards

The Cap IC – Q Exactive platform showed an outstanding separation and detection analytical sensitivity for polar metabolites. Metabolites were detected at 60 ppt (0.2 to 3.4 fmol on column), with S/N varying from 3-20. The LODs were in the range of 0.05 to 0.5 nmol/L. Isomeric compounds including: (a) α-D-glucose-1-phosphate, α-D-glucose-6-phosphate, and D-fructose-6-phosphate; (b) citrate and isocitrate; (c) trans- and cis-aconitate; and (d) fructose-1,6-phosphate and fructose-2,6-phosphate were baseline resolved. The resolution did not worsen even when the concentrations were lowered 10,000-fold, and the RT shifts were less than 0.04 min. HILIC using a ZIC-pHILIC column were performed afterwards on the same Q Exactive MS. UHPLC separation for the polar compounds was mostly unacceptable (data not shown), while HILIC showed good separation for most metabolites. However, the resolution on certain isomers like sugar phosphates and cis- and trans-aconitate were much worse compared to Cap IC. The analytical sensitivity by Cap IC system is generally 10-100 times better than the HILIC method.

FIGURE 1. Schematic diagram of capillary IC – Q Exactive platform for untargeted metabolic profiling

Data Analysis

Differential analyses were performed using Thermo Scientific™ SIEVE™ software version 2.1. Statistical results, putative IDs, and pathways were generated by searching ChemSpider® and KEGG™ databases. Metabolites of interest were identified via MS/MS mass spectral database matching. The raw data were converted to mzXML format using ProteoWizard and analyzed by XCMS Online and metaXCMS [2].

Peak # Metabolite Name Formula M-H On column (fmol) LOD

(S/N = 3,

nM)

1 D-Glucose C6H12O6 179.0561 0.17 0.3

2 Mevalonate C6H12O4 147.0663 2.0 0.1

3 Lactate C3H6O3 89.0244 3.4 0.1

4 Uridine C9H12N2O6 243.0623 1.2 0.25

5 α-D-Glucose 1-phosphate C6H13O9P 259.0224 1.2 0.2

6 α-D-glucose-6-phosphate C6H13O9P 259.0224 1.2 0.2

7 D-Fructose 6-phosphate C6H13O9P 259.0224 1.2 0.2

8 Adenosine 3'-5'-cyclic monophosphate (cAMP) C10H12N5O6P 328.0452 0.91 0.2

9 Tartrate C4H6O6 149.0092 2.0 0.5

10 2-oxoglutarate C5H6O5 145.0142 2.1 0.2

11 Adenosine 5'-monophosphate (AMP) C10H14N5O7P 346.0558 0.87 0.1

12 2-phosphoglycerate C3H7O7P 184.9857 1.6 0.3

13 Citrate C6H8O7 191.0197 1.6 0.2

14 Isocitrate C6H8O7 191.0197 1.6 0.05

15 cis-Aconitate C6H6O6 173.0092 1.7 0.2

16 trans-Aconitate C6H6O6 173.0092 1.7 0.2

17 Phosphoenolpyruvate C3H5O6P 166.9751 1.8 0.2

18 D-Fructose-1,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

19 D-Fructose-2,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

20 Dihydroxy acetone-Phosphate C3H7O6P 168.9908 1.8 0.04

21 Inosine 5'-monophosphate C10H13N4O8P 347.0398 0.87 0.1

FIGURE 2.Separation of 21 standard metabolites at 600 ppb by Cap IC (A), 60 ppt by Cap IC (B) and 600 ppb by HILIC method (C) with Orbitrap MS Injection volume 5 µL. Standard mixture for Cap IC was diluted in water; for HILIC, the mixture was diluted in acetonitrile/water (3:1, v/v).

FIGURE 3. Inter-day reproducibility analysis of the metabolite standards at 600 ppb by Cap IC with conductive detection (n=6). RSDs of intensity were 5.5%, 7.8%, and 6.0% and RSDs of RT were 6.5%, 8%, and 7.2 %, respectively, for three inorganic ions chloride, carbonate, and phosphate coexisting in the sample solution.

FIGURE 4. (A) The overlap of detected metabolite number in cell lysate by Cap IC and LC methods. (B) The overlap of aligned features among three pairs of cancer cell samples by metaXCMS analysis.

FIGURE 5. Separation of peaks corresponding to m/z 259.0224 in UM1 oral cancer cells by (A) Cap IC, (B) UHPLC, (C) Cap-LC, (D) ZIC-pHILIC, and (E) Cap-HILIC. The five methods were connected to the same Q Exactive MS.

Metabolic Profiling of Cell Lysates by Cap IC

We used 42 standards including isomeric compounds (corresponding to 36 m/z values) to evaluate the system performance. The same m/z list was used to extract the peaks from the UM1 cell sample from Cap IC, UHPLC, and HILIC analyses. As shown in Figure 4A, for this list of polar compounds Cap IC detected 65 peaks, ZIC-pHILIC detected 38 peaks, and UHPLC detected 29 peaks. A total of 26 peaks were detected by all three methods, but Cap IC showed much greater coverage. Cap IC detected 25 more peaks than HILIC with only one compound, acetyl-CoA, detected uniquely by HILIC. This is likely because the acetyl-CoA molecules are too large for this particular IC column to retain.

Differential Analysis, Pathway Mapping and Meta-Analysis

MetaXCMS software for second-order analysis of global metabolomic data was used to perform the meta-analysis.2 All three datasets were individually analyzed with XCMS Online, which had detected 11377, 13302, and 10532 aligned features for UM1, UM5, and NCSC sample groups, respectively. The results were combined and analyzed by metaXCMS. Figure 4B shows the significant features (P-value < 0.05, ratio > 2) in each sample set, and a total of 218 features were identified as the common ones for all three sample groups.

FIGURE 6. Chromatogram overlays of the 11 sugar monophosphates in three pairs of cancer cell samples. (A) Cancer stem cells (CSCs) versus non-stem cancer cells (NSCCs); (B) UM1 versus UM1-KD; (C) UM5 versus UM5-KD. The inserts in each figure are the peak overlays of the internal standard hippuric acid-d5 ([M-H]-183.0824) that was spiked in the cell lysate samples, which showed a very reproducible intensity.

Peak # Obs. [M-H]- Δ ppm Compound Identity Matching UM1/KD UM5/KD CSC / NSCC

RT MS/MS ratio P-value ratio p-value ratio P-value

1 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 0.49 0.002 0.38 <0.0001 0.98 0.9

2 259.0225 0.4 Unknown n/a + 1.7 0.07 1.8 0.46 1.4 0.003

3***

259.0225 0.5 α-D-Glucose 1-phosphate + +

2.5 0.01 1.8 0.008 4.5 <0.000

1

4 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 1.5 0.02 0.56 0.04 1.0 0.5

5

259.0226 0.7 D-Mannose 1-phosphate n/a +

2.4 <0.0001 1.6 <0.0001 0.67 <0.000

1

6 259.0225 0.5 D-myo-Inositol-4-phosphate n/a + 2.0 0.003 1.6 0.002 3.0 0.4

7

259.0226 0.8 α-D-Fructose 1-phosphate + +

1.1 0.3 0.56 0.0002 0.63 <0.000

1

8 259.0226 0.9 α-D-Galatose 1-phosphate + + 0.95 0.6 0.15 <0.0001 - -

9*** 259.0226 0.8 D-Fructose 6-phosphate + + 3.4 0.003 3.9 0.0001 16 0.0002

10*** 259.0226 0.8 α-D-Glucose 6-phosphate + + 3.2 0.001 3.3 0.009 11 0.0006

11*** 259.0226 0.9 D-Mannose 6-phosphate n/a + 1.8 0.07 2.1 0.004 4.0 0.0006

FIGURE 7. Cap IC/MS analysis revealed metabolic changes of the glycolysis pathway between oral CSCs and NSCCs. p: p-value, R: fold change

Table 2. Differential changes of 11 sugar monophosphates between three pairs of cellular samples. RT: retention time of standards; n/a: RT not available; *** represents those peaks sharing the same trends in multiple cell lines.

For Research use only. Not for use in diagnostic procedures. SeQuant and ZIC are registered trademarks of Merck Millipore International. Chempsider is a registered trademark of ChemZoo Inc. KEGG is a trademark of Kyoto University, Kanehisa Lab. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64131-EN 0614S

TABLE 1. Twenty-one metabolite standards used in Figure 2 and their LOD by Cap IC-Q Exactive MS

(A) (B) (C) (A) (B) (C)

4 Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry

Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry Junhua Wang1, Terri Christison1, Kaori Misuno2, Shen Hu2, Ralf Tautenhahn1, Linda Lopez1, Yingying Huang1 1Thermo Fisher Scientific, San Jose, CA; 2School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA

Conclusion • An untargeted metabolomic analysis of head and neck cancer cells has been

performed in this study.

• The outstanding resolution of IC has led to the differentiation of many isobaric and isomeric polar metabolites.

• Large-scale validation and in-depth integration of the metabolomics data with proteomics and transcriptomic data is to be conducted.

References 1. Lorenz, M.A.et al Anal. Chem., 2011, 83, 3406–3414

2. Tautenhahn, R. et al Anal. Chem., 2011, 83, 696-700

This work is published in Anal. Chem. DOI: 10.1021/ac500951v

Overview Purpose: Demonstrate Cap IC coupling with high-resolution, accurate-mass (HRAM) MS for untargeted metabolomics research analysis and the unique capability to differentiate isobaric metabolites.

Methods: A Thermo Scientific™ Dionex™ ICS-4000 capillary IC system coupled with a Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer for cell lysate detection and differential analysis.

Results: Metabolites were detected at low concentration 60 ppt (0.2 to 3.4 fmol on column) with S/N varying from 3-20. The LODs were in the range of 0.04 to 0.5 nmol/L. Outstanding separation for anionic polar metabolites and superior analytical sensitivities were achieved compared to reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC).

Introduction The metabolomics approach has obtained increasing attention in oral cancer study. A highly analytically sensitive research platform coupling capillary ion chromatography (Cap IC) with the Q Exactive mass spectrometer has been developed, which is applied to potential metabolic biomarkers profiling of oral squamous cell carcinoma (OSCC) metastasis from cell lysates. Cap IC has demonstrated outstanding separation for anionic polar metabolites and superior analytical sensitivities compared to RPLC and HILIC methods. Differential analysis finds significant changes in energy metabolism pathways, e.g., glycolysis cycle and tricarboxylic acid cycle. The experiments demonstrate Cap IC as a complementary separation technique by providing superior resolution and analytical sensitivity of polar metabolites combined with the high-resolution, accurate-mass measurement capabilities to differentiate isobaric metabolites.

Methods Sample Preparation

Three OSCC cell lines, UMSCC1, UMSCC5, cancer stem-like cells (CSC), and according wild-type controls with biological replicates were harvested and counted. Cellular metabolites were extracted using a liquid nitrogen snap-freezing method with methanol/water according the literature.1

Ion Chromatography and Liquid Chromatography

An ICS-4000 system with an electrolytic suppressor was used to convert the potassium hydroxide gradient to pure water. The Cap IC analysis was performed on a Thermo Scientific™ Dionex™ IonPac™ AS11HC-4µm, 0.4 x 250 mm column. The IC flow rate was 25 µL/min, supplemented post-column with 10 µL/min make-up flow of methanol/acetic acid (2 mM). The gradient started with 2 mM potassium hydroxide, rose to 12 mM at 13.5 min, then to 20 mM at 22.5 min and to 70 mM at 31.5 min. It was then held at 70 mM for 6 min, followed by a decrease to 2 mM in 0.1 min and held for 7.5 min to re-equilibrate the column. HILIC was performed on a 150 x 2.1 mm, 5 µm SeQuant®- ZIC®-pHILIC column at 250 µL/min. UHPLC on a Thermo Scientific™ Hypersil GOLD™ 150 x 2.1 mm, 1.9 µm column at 450 µL/min. LC was performed on a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC HPG system.

Mass Spectrometry

A Q Exactive quadrupole-Orbitrap mass spectrometer was operated under ESI negative mode for all detections. Full mass scan (m/z 67-1000) used resolving power 70,000 (at m/z 200) with automatic gain control (AGC) target of 1 x 106 ions and a maximum ion injection time (IT) of 50 ms. Data-dependent MS/MS was acquired on a “Top10” data-dependent mode using the following parameters: resolving power, 17,500; AGC, 1 x 105 ions; maximum IT, 100 ms; isolation window,1.5 amu; normalized collision energy, 35% ± 50%; underfill ratio, 1.0%. Source ionization parameters were optimized according to different flow rates. For all capillary flow IC, the settings were as follows: spray voltage, -2.8 kV; transfer capillary temperature, 300 ºC; S-lens level, 50; heater temperature, 125 ºC; sheath gas, 26; aux gas, 2. For higher flow LC, the following settings were used: spray voltage, -3.2 kV, heater temperature, 275 ºC; sheath gas,45; aux gas, 5.

Results Cap IC/MS Analysis of Metabolite Standards

The Cap IC – Q Exactive platform showed an outstanding separation and detection analytical sensitivity for polar metabolites. Metabolites were detected at 60 ppt (0.2 to 3.4 fmol on column), with S/N varying from 3-20. The LODs were in the range of 0.05 to 0.5 nmol/L. Isomeric compounds including: (a) α-D-glucose-1-phosphate, α-D-glucose-6-phosphate, and D-fructose-6-phosphate; (b) citrate and isocitrate; (c) trans- and cis-aconitate; and (d) fructose-1,6-phosphate and fructose-2,6-phosphate were baseline resolved. The resolution did not worsen even when the concentrations were lowered 10,000-fold, and the RT shifts were less than 0.04 min. HILIC using a ZIC-pHILIC column were performed afterwards on the same Q Exactive MS. UHPLC separation for the polar compounds was mostly unacceptable (data not shown), while HILIC showed good separation for most metabolites. However, the resolution on certain isomers like sugar phosphates and cis- and trans-aconitate were much worse compared to Cap IC. The analytical sensitivity by Cap IC system is generally 10-100 times better than the HILIC method.

FIGURE 1. Schematic diagram of capillary IC – Q Exactive platform for untargeted metabolic profiling

Data Analysis

Differential analyses were performed using Thermo Scientific™ SIEVE™ software version 2.1. Statistical results, putative IDs, and pathways were generated by searching ChemSpider® and KEGG™ databases. Metabolites of interest were identified via MS/MS mass spectral database matching. The raw data were converted to mzXML format using ProteoWizard and analyzed by XCMS Online and metaXCMS [2].

Peak # Metabolite Name Formula M-H On column (fmol) LOD

(S/N = 3,

nM)

1 D-Glucose C6H12O6 179.0561 0.17 0.3

2 Mevalonate C6H12O4 147.0663 2.0 0.1

3 Lactate C3H6O3 89.0244 3.4 0.1

4 Uridine C9H12N2O6 243.0623 1.2 0.25

5 α-D-Glucose 1-phosphate C6H13O9P 259.0224 1.2 0.2

6 α-D-glucose-6-phosphate C6H13O9P 259.0224 1.2 0.2

7 D-Fructose 6-phosphate C6H13O9P 259.0224 1.2 0.2

8 Adenosine 3'-5'-cyclic monophosphate (cAMP) C10H12N5O6P 328.0452 0.91 0.2

9 Tartrate C4H6O6 149.0092 2.0 0.5

10 2-oxoglutarate C5H6O5 145.0142 2.1 0.2

11 Adenosine 5'-monophosphate (AMP) C10H14N5O7P 346.0558 0.87 0.1

12 2-phosphoglycerate C3H7O7P 184.9857 1.6 0.3

13 Citrate C6H8O7 191.0197 1.6 0.2

14 Isocitrate C6H8O7 191.0197 1.6 0.05

15 cis-Aconitate C6H6O6 173.0092 1.7 0.2

16 trans-Aconitate C6H6O6 173.0092 1.7 0.2

17 Phosphoenolpyruvate C3H5O6P 166.9751 1.8 0.2

18 D-Fructose-1,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

19 D-Fructose-2,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

20 Dihydroxy acetone-Phosphate C3H7O6P 168.9908 1.8 0.04

21 Inosine 5'-monophosphate C10H13N4O8P 347.0398 0.87 0.1

FIGURE 2.Separation of 21 standard metabolites at 600 ppb by Cap IC (A), 60 ppt by Cap IC (B) and 600 ppb by HILIC method (C) with Orbitrap MS Injection volume 5 µL. Standard mixture for Cap IC was diluted in water; for HILIC, the mixture was diluted in acetonitrile/water (3:1, v/v).

FIGURE 3. Inter-day reproducibility analysis of the metabolite standards at 600 ppb by Cap IC with conductive detection (n=6). RSDs of intensity were 5.5%, 7.8%, and 6.0% and RSDs of RT were 6.5%, 8%, and 7.2 %, respectively, for three inorganic ions chloride, carbonate, and phosphate coexisting in the sample solution.

FIGURE 4. (A) The overlap of detected metabolite number in cell lysate by Cap IC and LC methods. (B) The overlap of aligned features among three pairs of cancer cell samples by metaXCMS analysis.

FIGURE 5. Separation of peaks corresponding to m/z 259.0224 in UM1 oral cancer cells by (A) Cap IC, (B) UHPLC, (C) Cap-LC, (D) ZIC-pHILIC, and (E) Cap-HILIC. The five methods were connected to the same Q Exactive MS.

Metabolic Profiling of Cell Lysates by Cap IC

We used 42 standards including isomeric compounds (corresponding to 36 m/z values) to evaluate the system performance. The same m/z list was used to extract the peaks from the UM1 cell sample from Cap IC, UHPLC, and HILIC analyses. As shown in Figure 4A, for this list of polar compounds Cap IC detected 65 peaks, ZIC-pHILIC detected 38 peaks, and UHPLC detected 29 peaks. A total of 26 peaks were detected by all three methods, but Cap IC showed much greater coverage. Cap IC detected 25 more peaks than HILIC with only one compound, acetyl-CoA, detected uniquely by HILIC. This is likely because the acetyl-CoA molecules are too large for this particular IC column to retain.

Differential Analysis, Pathway Mapping and Meta-Analysis

MetaXCMS software for second-order analysis of global metabolomic data was used to perform the meta-analysis.2 All three datasets were individually analyzed with XCMS Online, which had detected 11377, 13302, and 10532 aligned features for UM1, UM5, and NCSC sample groups, respectively. The results were combined and analyzed by metaXCMS. Figure 4B shows the significant features (P-value < 0.05, ratio > 2) in each sample set, and a total of 218 features were identified as the common ones for all three sample groups.

FIGURE 6. Chromatogram overlays of the 11 sugar monophosphates in three pairs of cancer cell samples. (A) Cancer stem cells (CSCs) versus non-stem cancer cells (NSCCs); (B) UM1 versus UM1-KD; (C) UM5 versus UM5-KD. The inserts in each figure are the peak overlays of the internal standard hippuric acid-d5 ([M-H]-183.0824) that was spiked in the cell lysate samples, which showed a very reproducible intensity.

Peak # Obs. [M-H]- Δ ppm Compound Identity Matching UM1/KD UM5/KD CSC / NSCC

RT MS/MS ratio P-value ratio p-value ratio P-value

1 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 0.49 0.002 0.38 <0.0001 0.98 0.9

2 259.0225 0.4 Unknown n/a + 1.7 0.07 1.8 0.46 1.4 0.003

3***

259.0225 0.5 α-D-Glucose 1-phosphate + +

2.5 0.01 1.8 0.008 4.5 <0.000

1

4 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 1.5 0.02 0.56 0.04 1.0 0.5

5

259.0226 0.7 D-Mannose 1-phosphate n/a +

2.4 <0.0001 1.6 <0.0001 0.67 <0.000

1

6 259.0225 0.5 D-myo-Inositol-4-phosphate n/a + 2.0 0.003 1.6 0.002 3.0 0.4

7

259.0226 0.8 α-D-Fructose 1-phosphate + +

1.1 0.3 0.56 0.0002 0.63 <0.000

1

8 259.0226 0.9 α-D-Galatose 1-phosphate + + 0.95 0.6 0.15 <0.0001 - -

9*** 259.0226 0.8 D-Fructose 6-phosphate + + 3.4 0.003 3.9 0.0001 16 0.0002

10*** 259.0226 0.8 α-D-Glucose 6-phosphate + + 3.2 0.001 3.3 0.009 11 0.0006

11*** 259.0226 0.9 D-Mannose 6-phosphate n/a + 1.8 0.07 2.1 0.004 4.0 0.0006

FIGURE 7. Cap IC/MS analysis revealed metabolic changes of the glycolysis pathway between oral CSCs and NSCCs. p: p-value, R: fold change

Table 2. Differential changes of 11 sugar monophosphates between three pairs of cellular samples. RT: retention time of standards; n/a: RT not available; *** represents those peaks sharing the same trends in multiple cell lines.

For Research use only. Not for use in diagnostic procedures. SeQuant and ZIC are registered trademarks of Merck Millipore International. Chempsider is a registered trademark of ChemZoo Inc. KEGG is a trademark of Kyoto University, Kanehisa Lab. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64131-EN 0614S

TABLE 1. Twenty-one metabolite standards used in Figure 2 and their LOD by Cap IC-Q Exactive MS

(A) (B) (C) (A) (B) (C)

5Thermo Scientific Poster Note • PN-64131-ASMS-EN-0614S

Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry Junhua Wang1, Terri Christison1, Kaori Misuno2, Shen Hu2, Ralf Tautenhahn1, Linda Lopez1, Yingying Huang1 1Thermo Fisher Scientific, San Jose, CA; 2School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA

Conclusion • An untargeted metabolomic analysis of head and neck cancer cells has been

performed in this study.

• The outstanding resolution of IC has led to the differentiation of many isobaric and isomeric polar metabolites.

• Large-scale validation and in-depth integration of the metabolomics data with proteomics and transcriptomic data is to be conducted.

References 1. Lorenz, M.A.et al Anal. Chem., 2011, 83, 3406–3414

2. Tautenhahn, R. et al Anal. Chem., 2011, 83, 696-700

This work is published in Anal. Chem. DOI: 10.1021/ac500951v

Overview Purpose: Demonstrate Cap IC coupling with high-resolution, accurate-mass (HRAM) MS for untargeted metabolomics research analysis and the unique capability to differentiate isobaric metabolites.

Methods: A Thermo Scientific™ Dionex™ ICS-4000 capillary IC system coupled with a Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer for cell lysate detection and differential analysis.

Results: Metabolites were detected at low concentration 60 ppt (0.2 to 3.4 fmol on column) with S/N varying from 3-20. The LODs were in the range of 0.04 to 0.5 nmol/L. Outstanding separation for anionic polar metabolites and superior analytical sensitivities were achieved compared to reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC).

Introduction The metabolomics approach has obtained increasing attention in oral cancer study. A highly analytically sensitive research platform coupling capillary ion chromatography (Cap IC) with the Q Exactive mass spectrometer has been developed, which is applied to potential metabolic biomarkers profiling of oral squamous cell carcinoma (OSCC) metastasis from cell lysates. Cap IC has demonstrated outstanding separation for anionic polar metabolites and superior analytical sensitivities compared to RPLC and HILIC methods. Differential analysis finds significant changes in energy metabolism pathways, e.g., glycolysis cycle and tricarboxylic acid cycle. The experiments demonstrate Cap IC as a complementary separation technique by providing superior resolution and analytical sensitivity of polar metabolites combined with the high-resolution, accurate-mass measurement capabilities to differentiate isobaric metabolites.

Methods Sample Preparation

Three OSCC cell lines, UMSCC1, UMSCC5, cancer stem-like cells (CSC), and according wild-type controls with biological replicates were harvested and counted. Cellular metabolites were extracted using a liquid nitrogen snap-freezing method with methanol/water according the literature.1

Ion Chromatography and Liquid Chromatography

An ICS-4000 system with an electrolytic suppressor was used to convert the potassium hydroxide gradient to pure water. The Cap IC analysis was performed on a Thermo Scientific™ Dionex™ IonPac™ AS11HC-4µm, 0.4 x 250 mm column. The IC flow rate was 25 µL/min, supplemented post-column with 10 µL/min make-up flow of methanol/acetic acid (2 mM). The gradient started with 2 mM potassium hydroxide, rose to 12 mM at 13.5 min, then to 20 mM at 22.5 min and to 70 mM at 31.5 min. It was then held at 70 mM for 6 min, followed by a decrease to 2 mM in 0.1 min and held for 7.5 min to re-equilibrate the column. HILIC was performed on a 150 x 2.1 mm, 5 µm SeQuant®- ZIC®-pHILIC column at 250 µL/min. UHPLC on a Thermo Scientific™ Hypersil GOLD™ 150 x 2.1 mm, 1.9 µm column at 450 µL/min. LC was performed on a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC HPG system.

Mass Spectrometry

A Q Exactive quadrupole-Orbitrap mass spectrometer was operated under ESI negative mode for all detections. Full mass scan (m/z 67-1000) used resolving power 70,000 (at m/z 200) with automatic gain control (AGC) target of 1 x 106 ions and a maximum ion injection time (IT) of 50 ms. Data-dependent MS/MS was acquired on a “Top10” data-dependent mode using the following parameters: resolving power, 17,500; AGC, 1 x 105 ions; maximum IT, 100 ms; isolation window,1.5 amu; normalized collision energy, 35% ± 50%; underfill ratio, 1.0%. Source ionization parameters were optimized according to different flow rates. For all capillary flow IC, the settings were as follows: spray voltage, -2.8 kV; transfer capillary temperature, 300 ºC; S-lens level, 50; heater temperature, 125 ºC; sheath gas, 26; aux gas, 2. For higher flow LC, the following settings were used: spray voltage, -3.2 kV, heater temperature, 275 ºC; sheath gas,45; aux gas, 5.

Results Cap IC/MS Analysis of Metabolite Standards

The Cap IC – Q Exactive platform showed an outstanding separation and detection analytical sensitivity for polar metabolites. Metabolites were detected at 60 ppt (0.2 to 3.4 fmol on column), with S/N varying from 3-20. The LODs were in the range of 0.05 to 0.5 nmol/L. Isomeric compounds including: (a) α-D-glucose-1-phosphate, α-D-glucose-6-phosphate, and D-fructose-6-phosphate; (b) citrate and isocitrate; (c) trans- and cis-aconitate; and (d) fructose-1,6-phosphate and fructose-2,6-phosphate were baseline resolved. The resolution did not worsen even when the concentrations were lowered 10,000-fold, and the RT shifts were less than 0.04 min. HILIC using a ZIC-pHILIC column were performed afterwards on the same Q Exactive MS. UHPLC separation for the polar compounds was mostly unacceptable (data not shown), while HILIC showed good separation for most metabolites. However, the resolution on certain isomers like sugar phosphates and cis- and trans-aconitate were much worse compared to Cap IC. The analytical sensitivity by Cap IC system is generally 10-100 times better than the HILIC method.

FIGURE 1. Schematic diagram of capillary IC – Q Exactive platform for untargeted metabolic profiling

Data Analysis

Differential analyses were performed using Thermo Scientific™ SIEVE™ software version 2.1. Statistical results, putative IDs, and pathways were generated by searching ChemSpider® and KEGG™ databases. Metabolites of interest were identified via MS/MS mass spectral database matching. The raw data were converted to mzXML format using ProteoWizard and analyzed by XCMS Online and metaXCMS [2].

Peak # Metabolite Name Formula M-H On column (fmol) LOD

(S/N = 3,

nM)

1 D-Glucose C6H12O6 179.0561 0.17 0.3

2 Mevalonate C6H12O4 147.0663 2.0 0.1

3 Lactate C3H6O3 89.0244 3.4 0.1

4 Uridine C9H12N2O6 243.0623 1.2 0.25

5 α-D-Glucose 1-phosphate C6H13O9P 259.0224 1.2 0.2

6 α-D-glucose-6-phosphate C6H13O9P 259.0224 1.2 0.2

7 D-Fructose 6-phosphate C6H13O9P 259.0224 1.2 0.2

8 Adenosine 3'-5'-cyclic monophosphate (cAMP) C10H12N5O6P 328.0452 0.91 0.2

9 Tartrate C4H6O6 149.0092 2.0 0.5

10 2-oxoglutarate C5H6O5 145.0142 2.1 0.2

11 Adenosine 5'-monophosphate (AMP) C10H14N5O7P 346.0558 0.87 0.1

12 2-phosphoglycerate C3H7O7P 184.9857 1.6 0.3

13 Citrate C6H8O7 191.0197 1.6 0.2

14 Isocitrate C6H8O7 191.0197 1.6 0.05

15 cis-Aconitate C6H6O6 173.0092 1.7 0.2

16 trans-Aconitate C6H6O6 173.0092 1.7 0.2

17 Phosphoenolpyruvate C3H5O6P 166.9751 1.8 0.2

18 D-Fructose-1,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

19 D-Fructose-2,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

20 Dihydroxy acetone-Phosphate C3H7O6P 168.9908 1.8 0.04

21 Inosine 5'-monophosphate C10H13N4O8P 347.0398 0.87 0.1

FIGURE 2.Separation of 21 standard metabolites at 600 ppb by Cap IC (A), 60 ppt by Cap IC (B) and 600 ppb by HILIC method (C) with Orbitrap MS Injection volume 5 µL. Standard mixture for Cap IC was diluted in water; for HILIC, the mixture was diluted in acetonitrile/water (3:1, v/v).

FIGURE 3. Inter-day reproducibility analysis of the metabolite standards at 600 ppb by Cap IC with conductive detection (n=6). RSDs of intensity were 5.5%, 7.8%, and 6.0% and RSDs of RT were 6.5%, 8%, and 7.2 %, respectively, for three inorganic ions chloride, carbonate, and phosphate coexisting in the sample solution.

FIGURE 4. (A) The overlap of detected metabolite number in cell lysate by Cap IC and LC methods. (B) The overlap of aligned features among three pairs of cancer cell samples by metaXCMS analysis.

FIGURE 5. Separation of peaks corresponding to m/z 259.0224 in UM1 oral cancer cells by (A) Cap IC, (B) UHPLC, (C) Cap-LC, (D) ZIC-pHILIC, and (E) Cap-HILIC. The five methods were connected to the same Q Exactive MS.

Metabolic Profiling of Cell Lysates by Cap IC

We used 42 standards including isomeric compounds (corresponding to 36 m/z values) to evaluate the system performance. The same m/z list was used to extract the peaks from the UM1 cell sample from Cap IC, UHPLC, and HILIC analyses. As shown in Figure 4A, for this list of polar compounds Cap IC detected 65 peaks, ZIC-pHILIC detected 38 peaks, and UHPLC detected 29 peaks. A total of 26 peaks were detected by all three methods, but Cap IC showed much greater coverage. Cap IC detected 25 more peaks than HILIC with only one compound, acetyl-CoA, detected uniquely by HILIC. This is likely because the acetyl-CoA molecules are too large for this particular IC column to retain.

Differential Analysis, Pathway Mapping and Meta-Analysis

MetaXCMS software for second-order analysis of global metabolomic data was used to perform the meta-analysis.2 All three datasets were individually analyzed with XCMS Online, which had detected 11377, 13302, and 10532 aligned features for UM1, UM5, and NCSC sample groups, respectively. The results were combined and analyzed by metaXCMS. Figure 4B shows the significant features (P-value < 0.05, ratio > 2) in each sample set, and a total of 218 features were identified as the common ones for all three sample groups.

FIGURE 6. Chromatogram overlays of the 11 sugar monophosphates in three pairs of cancer cell samples. (A) Cancer stem cells (CSCs) versus non-stem cancer cells (NSCCs); (B) UM1 versus UM1-KD; (C) UM5 versus UM5-KD. The inserts in each figure are the peak overlays of the internal standard hippuric acid-d5 ([M-H]-183.0824) that was spiked in the cell lysate samples, which showed a very reproducible intensity.

Peak # Obs. [M-H]- Δ ppm Compound Identity Matching UM1/KD UM5/KD CSC / NSCC

RT MS/MS ratio P-value ratio p-value ratio P-value

1 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 0.49 0.002 0.38 <0.0001 0.98 0.9

2 259.0225 0.4 Unknown n/a + 1.7 0.07 1.8 0.46 1.4 0.003

3***

259.0225 0.5 α-D-Glucose 1-phosphate + +

2.5 0.01 1.8 0.008 4.5 <0.000

1

4 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 1.5 0.02 0.56 0.04 1.0 0.5

5

259.0226 0.7 D-Mannose 1-phosphate n/a +

2.4 <0.0001 1.6 <0.0001 0.67 <0.000

1

6 259.0225 0.5 D-myo-Inositol-4-phosphate n/a + 2.0 0.003 1.6 0.002 3.0 0.4

7

259.0226 0.8 α-D-Fructose 1-phosphate + +

1.1 0.3 0.56 0.0002 0.63 <0.000

1

8 259.0226 0.9 α-D-Galatose 1-phosphate + + 0.95 0.6 0.15 <0.0001 - -

9*** 259.0226 0.8 D-Fructose 6-phosphate + + 3.4 0.003 3.9 0.0001 16 0.0002

10*** 259.0226 0.8 α-D-Glucose 6-phosphate + + 3.2 0.001 3.3 0.009 11 0.0006

11*** 259.0226 0.9 D-Mannose 6-phosphate n/a + 1.8 0.07 2.1 0.004 4.0 0.0006

FIGURE 7. Cap IC/MS analysis revealed metabolic changes of the glycolysis pathway between oral CSCs and NSCCs. p: p-value, R: fold change

Table 2. Differential changes of 11 sugar monophosphates between three pairs of cellular samples. RT: retention time of standards; n/a: RT not available; *** represents those peaks sharing the same trends in multiple cell lines.

For Research use only. Not for use in diagnostic procedures. SeQuant and ZIC are registered trademarks of Merck Millipore International. Chempsider is a registered trademark of ChemZoo Inc. KEGG is a trademark of Kyoto University, Kanehisa Lab. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64131-EN 0614S

TABLE 1. Twenty-one metabolite standards used in Figure 2 and their LOD by Cap IC-Q Exactive MS

(A) (B) (C) (A) (B) (C)

6 Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry

Metabolomic Profiling of Anionic Metabolites in Oral Cancer Cells by Capillary Ion Chromatography HRAM Mass Spectrometry Junhua Wang1, Terri Christison1, Kaori Misuno2, Shen Hu2, Ralf Tautenhahn1, Linda Lopez1, Yingying Huang1 1Thermo Fisher Scientific, San Jose, CA; 2School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA

Conclusion • An untargeted metabolomic analysis of head and neck cancer cells has been

performed in this study.

• The outstanding resolution of IC has led to the differentiation of many isobaric and isomeric polar metabolites.

• Large-scale validation and in-depth integration of the metabolomics data with proteomics and transcriptomic data is to be conducted.

References 1. Lorenz, M.A.et al Anal. Chem., 2011, 83, 3406–3414

2. Tautenhahn, R. et al Anal. Chem., 2011, 83, 696-700

This work is published in Anal. Chem. DOI: 10.1021/ac500951v

Overview Purpose: Demonstrate Cap IC coupling with high-resolution, accurate-mass (HRAM) MS for untargeted metabolomics research analysis and the unique capability to differentiate isobaric metabolites.

Methods: A Thermo Scientific™ Dionex™ ICS-4000 capillary IC system coupled with a Thermo Scientific™ Q Exactive™ hybrid quadrupole-Orbitrap mass spectrometer for cell lysate detection and differential analysis.

Results: Metabolites were detected at low concentration 60 ppt (0.2 to 3.4 fmol on column) with S/N varying from 3-20. The LODs were in the range of 0.04 to 0.5 nmol/L. Outstanding separation for anionic polar metabolites and superior analytical sensitivities were achieved compared to reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC).

Introduction The metabolomics approach has obtained increasing attention in oral cancer study. A highly analytically sensitive research platform coupling capillary ion chromatography (Cap IC) with the Q Exactive mass spectrometer has been developed, which is applied to potential metabolic biomarkers profiling of oral squamous cell carcinoma (OSCC) metastasis from cell lysates. Cap IC has demonstrated outstanding separation for anionic polar metabolites and superior analytical sensitivities compared to RPLC and HILIC methods. Differential analysis finds significant changes in energy metabolism pathways, e.g., glycolysis cycle and tricarboxylic acid cycle. The experiments demonstrate Cap IC as a complementary separation technique by providing superior resolution and analytical sensitivity of polar metabolites combined with the high-resolution, accurate-mass measurement capabilities to differentiate isobaric metabolites.

Methods Sample Preparation

Three OSCC cell lines, UMSCC1, UMSCC5, cancer stem-like cells (CSC), and according wild-type controls with biological replicates were harvested and counted. Cellular metabolites were extracted using a liquid nitrogen snap-freezing method with methanol/water according the literature.1

Ion Chromatography and Liquid Chromatography

An ICS-4000 system with an electrolytic suppressor was used to convert the potassium hydroxide gradient to pure water. The Cap IC analysis was performed on a Thermo Scientific™ Dionex™ IonPac™ AS11HC-4µm, 0.4 x 250 mm column. The IC flow rate was 25 µL/min, supplemented post-column with 10 µL/min make-up flow of methanol/acetic acid (2 mM). The gradient started with 2 mM potassium hydroxide, rose to 12 mM at 13.5 min, then to 20 mM at 22.5 min and to 70 mM at 31.5 min. It was then held at 70 mM for 6 min, followed by a decrease to 2 mM in 0.1 min and held for 7.5 min to re-equilibrate the column. HILIC was performed on a 150 x 2.1 mm, 5 µm SeQuant®- ZIC®-pHILIC column at 250 µL/min. UHPLC on a Thermo Scientific™ Hypersil GOLD™ 150 x 2.1 mm, 1.9 µm column at 450 µL/min. LC was performed on a Thermo Scientific™ Dionex™ UltiMate™ 3000 RSLC HPG system.

Mass Spectrometry

A Q Exactive quadrupole-Orbitrap mass spectrometer was operated under ESI negative mode for all detections. Full mass scan (m/z 67-1000) used resolving power 70,000 (at m/z 200) with automatic gain control (AGC) target of 1 x 106 ions and a maximum ion injection time (IT) of 50 ms. Data-dependent MS/MS was acquired on a “Top10” data-dependent mode using the following parameters: resolving power, 17,500; AGC, 1 x 105 ions; maximum IT, 100 ms; isolation window,1.5 amu; normalized collision energy, 35% ± 50%; underfill ratio, 1.0%. Source ionization parameters were optimized according to different flow rates. For all capillary flow IC, the settings were as follows: spray voltage, -2.8 kV; transfer capillary temperature, 300 ºC; S-lens level, 50; heater temperature, 125 ºC; sheath gas, 26; aux gas, 2. For higher flow LC, the following settings were used: spray voltage, -3.2 kV, heater temperature, 275 ºC; sheath gas,45; aux gas, 5.

Results Cap IC/MS Analysis of Metabolite Standards

The Cap IC – Q Exactive platform showed an outstanding separation and detection analytical sensitivity for polar metabolites. Metabolites were detected at 60 ppt (0.2 to 3.4 fmol on column), with S/N varying from 3-20. The LODs were in the range of 0.05 to 0.5 nmol/L. Isomeric compounds including: (a) α-D-glucose-1-phosphate, α-D-glucose-6-phosphate, and D-fructose-6-phosphate; (b) citrate and isocitrate; (c) trans- and cis-aconitate; and (d) fructose-1,6-phosphate and fructose-2,6-phosphate were baseline resolved. The resolution did not worsen even when the concentrations were lowered 10,000-fold, and the RT shifts were less than 0.04 min. HILIC using a ZIC-pHILIC column were performed afterwards on the same Q Exactive MS. UHPLC separation for the polar compounds was mostly unacceptable (data not shown), while HILIC showed good separation for most metabolites. However, the resolution on certain isomers like sugar phosphates and cis- and trans-aconitate were much worse compared to Cap IC. The analytical sensitivity by Cap IC system is generally 10-100 times better than the HILIC method.

FIGURE 1. Schematic diagram of capillary IC – Q Exactive platform for untargeted metabolic profiling

Data Analysis

Differential analyses were performed using Thermo Scientific™ SIEVE™ software version 2.1. Statistical results, putative IDs, and pathways were generated by searching ChemSpider® and KEGG™ databases. Metabolites of interest were identified via MS/MS mass spectral database matching. The raw data were converted to mzXML format using ProteoWizard and analyzed by XCMS Online and metaXCMS [2].

Peak # Metabolite Name Formula M-H On column (fmol) LOD

(S/N = 3,

nM)

1 D-Glucose C6H12O6 179.0561 0.17 0.3

2 Mevalonate C6H12O4 147.0663 2.0 0.1

3 Lactate C3H6O3 89.0244 3.4 0.1

4 Uridine C9H12N2O6 243.0623 1.2 0.25

5 α-D-Glucose 1-phosphate C6H13O9P 259.0224 1.2 0.2

6 α-D-glucose-6-phosphate C6H13O9P 259.0224 1.2 0.2

7 D-Fructose 6-phosphate C6H13O9P 259.0224 1.2 0.2

8 Adenosine 3'-5'-cyclic monophosphate (cAMP) C10H12N5O6P 328.0452 0.91 0.2

9 Tartrate C4H6O6 149.0092 2.0 0.5

10 2-oxoglutarate C5H6O5 145.0142 2.1 0.2

11 Adenosine 5'-monophosphate (AMP) C10H14N5O7P 346.0558 0.87 0.1

12 2-phosphoglycerate C3H7O7P 184.9857 1.6 0.3

13 Citrate C6H8O7 191.0197 1.6 0.2

14 Isocitrate C6H8O7 191.0197 1.6 0.05

15 cis-Aconitate C6H6O6 173.0092 1.7 0.2

16 trans-Aconitate C6H6O6 173.0092 1.7 0.2

17 Phosphoenolpyruvate C3H5O6P 166.9751 1.8 0.2

18 D-Fructose-1,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

19 D-Fructose-2,6-diphosphate C6H14O12P2 338.9888 0.88 0.1

20 Dihydroxy acetone-Phosphate C3H7O6P 168.9908 1.8 0.04

21 Inosine 5'-monophosphate C10H13N4O8P 347.0398 0.87 0.1

FIGURE 2.Separation of 21 standard metabolites at 600 ppb by Cap IC (A), 60 ppt by Cap IC (B) and 600 ppb by HILIC method (C) with Orbitrap MS Injection volume 5 µL. Standard mixture for Cap IC was diluted in water; for HILIC, the mixture was diluted in acetonitrile/water (3:1, v/v).

FIGURE 3. Inter-day reproducibility analysis of the metabolite standards at 600 ppb by Cap IC with conductive detection (n=6). RSDs of intensity were 5.5%, 7.8%, and 6.0% and RSDs of RT were 6.5%, 8%, and 7.2 %, respectively, for three inorganic ions chloride, carbonate, and phosphate coexisting in the sample solution.

FIGURE 4. (A) The overlap of detected metabolite number in cell lysate by Cap IC and LC methods. (B) The overlap of aligned features among three pairs of cancer cell samples by metaXCMS analysis.

FIGURE 5. Separation of peaks corresponding to m/z 259.0224 in UM1 oral cancer cells by (A) Cap IC, (B) UHPLC, (C) Cap-LC, (D) ZIC-pHILIC, and (E) Cap-HILIC. The five methods were connected to the same Q Exactive MS.

Metabolic Profiling of Cell Lysates by Cap IC

We used 42 standards including isomeric compounds (corresponding to 36 m/z values) to evaluate the system performance. The same m/z list was used to extract the peaks from the UM1 cell sample from Cap IC, UHPLC, and HILIC analyses. As shown in Figure 4A, for this list of polar compounds Cap IC detected 65 peaks, ZIC-pHILIC detected 38 peaks, and UHPLC detected 29 peaks. A total of 26 peaks were detected by all three methods, but Cap IC showed much greater coverage. Cap IC detected 25 more peaks than HILIC with only one compound, acetyl-CoA, detected uniquely by HILIC. This is likely because the acetyl-CoA molecules are too large for this particular IC column to retain.

Differential Analysis, Pathway Mapping and Meta-Analysis

MetaXCMS software for second-order analysis of global metabolomic data was used to perform the meta-analysis.2 All three datasets were individually analyzed with XCMS Online, which had detected 11377, 13302, and 10532 aligned features for UM1, UM5, and NCSC sample groups, respectively. The results were combined and analyzed by metaXCMS. Figure 4B shows the significant features (P-value < 0.05, ratio > 2) in each sample set, and a total of 218 features were identified as the common ones for all three sample groups.

FIGURE 6. Chromatogram overlays of the 11 sugar monophosphates in three pairs of cancer cell samples. (A) Cancer stem cells (CSCs) versus non-stem cancer cells (NSCCs); (B) UM1 versus UM1-KD; (C) UM5 versus UM5-KD. The inserts in each figure are the peak overlays of the internal standard hippuric acid-d5 ([M-H]-183.0824) that was spiked in the cell lysate samples, which showed a very reproducible intensity.

Peak # Obs. [M-H]- Δ ppm Compound Identity Matching UM1/KD UM5/KD CSC / NSCC

RT MS/MS ratio P-value ratio p-value ratio P-value

1 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 0.49 0.002 0.38 <0.0001 0.98 0.9

2 259.0225 0.4 Unknown n/a + 1.7 0.07 1.8 0.46 1.4 0.003

3***

259.0225 0.5 α-D-Glucose 1-phosphate + +

2.5 0.01 1.8 0.008 4.5 <0.000

1

4 259.0225 0.4 D-myo-Inositol (1 or 3)-phosphate n/a + 1.5 0.02 0.56 0.04 1.0 0.5

5

259.0226 0.7 D-Mannose 1-phosphate n/a +

2.4 <0.0001 1.6 <0.0001 0.67 <0.000

1

6 259.0225 0.5 D-myo-Inositol-4-phosphate n/a + 2.0 0.003 1.6 0.002 3.0 0.4

7

259.0226 0.8 α-D-Fructose 1-phosphate + +

1.1 0.3 0.56 0.0002 0.63 <0.000

1

8 259.0226 0.9 α-D-Galatose 1-phosphate + + 0.95 0.6 0.15 <0.0001 - -

9*** 259.0226 0.8 D-Fructose 6-phosphate + + 3.4 0.003 3.9 0.0001 16 0.0002

10*** 259.0226 0.8 α-D-Glucose 6-phosphate + + 3.2 0.001 3.3 0.009 11 0.0006

11*** 259.0226 0.9 D-Mannose 6-phosphate n/a + 1.8 0.07 2.1 0.004 4.0 0.0006

FIGURE 7. Cap IC/MS analysis revealed metabolic changes of the glycolysis pathway between oral CSCs and NSCCs. p: p-value, R: fold change

Table 2. Differential changes of 11 sugar monophosphates between three pairs of cellular samples. RT: retention time of standards; n/a: RT not available; *** represents those peaks sharing the same trends in multiple cell lines.

For Research use only. Not for use in diagnostic procedures. SeQuant and ZIC are registered trademarks of Merck Millipore International. Chempsider is a registered trademark of ChemZoo Inc. KEGG is a trademark of Kyoto University, Kanehisa Lab. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO64131-EN 0614S

TABLE 1. Twenty-one metabolite standards used in Figure 2 and their LOD by Cap IC-Q Exactive MS

(A) (B) (C) (A) (B) (C)

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