Article pubs.acs.org/ac
Targeted Metabolomic Analysis of Head and Neck Cancer Cells Using High Performance Ion Chromatography Coupled with a Q Exactive HF Mass Spectrometer Shen Hu,*,† Junhua Wang,*,‡ Eoon Hye Ji,† Terri Christison,‡ Linda Lopez,‡ and Yingying Huang‡ †
School of Dentistry and Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, United States ‡ Thermo Fisher Scientific Inc, San Jose, California 95134, United States S Supporting Information *
ABSTRACT: In this study, we have demonstrated a targeted metabolomics method for analysis of cancer cells, based on high-performance ion chromatography (IC) separation, Q Exactive HF MS for high-resolution and accurate-mass (HR/AM) measurement and the use of stable isotope-labeled internal standards for absolute quantitation. Our method offers great technical advantages for metabolite analysis, including exquisite sensitivity, high speed and reproducibility, and wide dynamic range. The high-performance IC provided fast separation of cellular metabolites within 20 min and excellent resolving power for polar molecules including many isobaric metabolites. The IC/Q Exactive HF MS achieved wide dynamic ranges of 5 orders of magnitude for six targeted metabolites, pyruvate, succinic acid, malic acid, citric acid, fumaric acid, and alphaketoglutaric acid, with R2 ≈ 0.99. Using this platform, metabolites can be simultaneously quantified from low fmol/μL to nmol/μL levels in cellular samples. The high flow rate IC at 380 μL/min has shown excellent reproducibility for a large set of samples (150 injections), with minimal variations of retention time (SD < ± 0.03 min). In addition, the IC-MS-based approach acquires targeted and global metabolomic data in a same analytical run, and the use of stable isotope-labeled standards facilitates accurate quantitation of targeted metabolites in large-scale metabolomics analysis. This metabolomics approach has been successfully applied to analysis of targeted metabolites in head and neck cancer cells as well as cancer stem-like cells (CSCs), and the findings indicate that the metabolic phenotypes may be distinct between high and low invasive head and neck cancer cells and between CSCs and non-SCCs.
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drates, organic acids, sugar phosphates, and nucleotides in biological samples.10−12 Recently, a high-performance analytical platform by coupling capillary ion chromatography (CapIC) with Q Exactive mass spectrometer has been successfully developed for global metabolic analysis of head and neck cancer cells.9 The outstanding resolution of CapIC has led to the differentiation of many isobaric and isomeric polar metabolites, and the method has shown a broad coverage of glycolytic and tricarboxylic acid cycle (TCA) intermediates. Significant alterations in TCA and glycolytic metabolites in cancer stem cells versus nonstem cancer cells were also observed.9 Targeted metabolomics is a quantitative approach wherein a set of known targeted metabolites are quantified based on their relative abundance when comparing to internal or external reference standards.1,2 Isotopically labeled standards are ideal external calibration references to spike into a metabolomic sample for targeted quantitative analysis in the liquid
etabolomics aims to measure a wide breadth of small molecules (metabolome) in the context of physiological stimuli or disease states.1 With the newly evolved mass spectrometry (MS) and nuclear magnetic resonance spectroscopy methods, metabolomics has now become a valuable approach for profiling of disease samples to identify biomarkers for diagnosis, prognosis, or treatment efficacy.2,3 The general problems encountered when performing a metabolomic analysis are the highly complex nature and the wide concentration dynamic range of the compounds present in a metabolome. Separation science plays an important role in metabolomics by reducing the sample complexity to achieve a comprehensive profiling analysis.4 The strength of mass spectrometry (MS)-based metabolomics is best realized when coupled to a separation technique, such as capillary electrophoresis,5 gas chromatography (GC),6 or liquid chromatography (LC).7 Ion chromatography (IC) or ion-exchange chromatography offers an excellent complementary platform for separation of charged and polar compounds.8,9 With its unique selectivity, researchers have attempted to couple IC with MS for targeted screening and quantification of metabolites such as carbohy© XXXX American Chemical Society
Received: April 10, 2015 Accepted: May 14, 2015
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DOI: 10.1021/acs.analchem.5b01350 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
Figure 1. (A) Analysis of six stable isotope-labeled standards by IC with Q Exactive HF MS. (B) These standard compounds include pyruvate and 5 intermediates (succinic acid, malic acid, citric acid, fumaric acid, and alpha-ketoglutaric acid) of tricarboxylic acid cycle.
chromatography−mass spectrometry (LC−MS) experiment because of their similar ionization effect and chromatographic retention. The resulting data of targeted metabolomics can then
be used for pathway analysis or as input variables for statistical analysis. Because of reliable measurements of metabolites among complex sample matrix, targeted metabolomics can B
DOI: 10.1021/acs.analchem.5b01350 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
μL size loop was used for sample injection, and the separation was performed using a Thermo Scientific Dionex IonPac AS11HC 2 × 250 mm, 4 μm particle size column. IC flow rate was 0.38 mL/min supplemented postcolumn with 0.060 mL/min makeup flow of MeOH/2 mM HOAc (Figure 1 of the Supporting Information). The gradient conditions for IC separation are shown in Figure 2 of the Supporting Information. Mass Spectrometry. The Q Exactive HF mass spectrometer was operated under an ESI negative mode for all detections. Full mass scan (m/z 67−1000) was used at a resolution of 120000 with a scan rate at ∼3.5 Hz. The automatic gain control (AGC) target was set at 1 × 106 ions, and maximum ion injection time (IT) was at 100 ms. Source ionization parameters were optimized with the spray voltage at 3.5 kV, and other parameters were as follows: transfer temperature at 320 °C; S-Lens level at 50; heater temperature at 325 °C; Sheath gas at 36 and Aux gas at 5. Data Processing. Data were acquired in full scan mode. Differential analysis of profiling data was performed using the Thermo Scientific SIEVE 2.2 software. Qualitative and quantitative analysis of targeted compounds was performed using the Thermo Scientific Tracefinder 3.2 software according to the manufacturer’s manual. Briefly, the six isotope-labeled compounds were entered into the Tracefinder local compound database and linked to the targeted compounds. Calibration curves were then established based on the isotope-labeled standards to determine the quantity of the targeted compounds.
provide accurate information about the dynamics and fluxes of metabolites. In this study, a high flow rate IC system with Q Exactive HF MS was demonstrated for targeted analysis of metabolites in head and neck cancer cells and validation of our previous findings obtained by global metabolomic analysis with the CapIC-MS method.9 Six stable isotope-labeled standards for pyruvate and five TCA intermediates were used to establish the calibration curves for targeted quantitative analysis of these metabolites in multiple cancer cell lines as well as cancer stemlike cells (CSCs). By using the Thermo Scientific Tracefinder software, the endogenous levels of the six targeted metabolites in a large set of metabolic samples were quantified quickly and accurately.
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EXPERIMENTAL SECTION Sample Preparation. A spherogenic assay was used to enrich and isolate stem-like oral cancer cells (CSCs) and nonstem cancer cells (NSCCs) from cultured UM1 cancer cells, as described in our previously published study.13 UM1, UM2, UM5, and UM6 head and neck cancer cells were cultured in Dulbecco’s modified eagle medium (DMEM) plus 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen, Carlsbad, CA). The cells were maintained at 37 °C in a humidified 5% CO2 incubator and passaged when they reached 90−95% confluence. Cell numbers were counted with a Cell Viability Analyzer (Beckman, Brea CA). Cellular metabolites were extracted using the liquid nitrogen (LN2) snap-freezing method with methanol/water as described previously.14 We used the similar extraction method to prepare metabolome samples from cancer cells in our global metabolomics studies.9 In brief, the cells were quickly washed twice with ice-cold phosphate-buffered saline (PBS) in a cold room to remove medium components and then quickly rinsed with water. After removal of water, the cells were flash frozen with LN2 and 1.0 mL of ice cold 90% MeOH; CHCl3 was immediately added to each plate, and cells were scraped/ suspended with a cell scraper. Extracts were transferred to microcentrifuge tubes and pelleted at 4 °C for 3 min at 16100g. Supernatants were then transferred to new microcentrifuge tubes for IC−MS analysis. All experiments were performed in 3 to 12 replicates. Stable isotope-labeled internal standards were supplied by the Cambridge Isotope Laboratories (Tewksbury, MA). Six stable isotope labeled standards, including pyruvate (13C3, 99%), malic acid (13C4, 99%), fumaric acid (13C4, 99%), succinic acid (13C4, 99%), alpha-ketoglutaric acid (13C5, 99%), and citric acid (2,2,4,4-D4, 98%), were pooled at the same concentrations and then serially diluted, ranging from 10000, 5000, 1000, 500, 100, 50, 10, 5, 1, 0.5, and 0.1 pg/μL (a total of 11 serial dilutions). The 11 serially diluted standard mixtures were randomly spiked into the cellular metabolomic samples at least in triplicates for absolute quantification of targeted metabolites in cancer cells. Ion Chromatography. A Dionex ICS-5000+ HPIC ion chromatography was coupled with Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer for the targeted metabolomic analysis (Thermo Scientific, San Jose, CA). The IC was equipped with an anion electrolytic suppressor (Thermo Scientific Dionex AERS 500) to convert the potassium hydroxide gradient into pure water before the sample enters the mass spectrometer. A 2 μL partial loop on a 5
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RESULTS AND DISCUSSION High-Performance IC for Metabolite Analysis. A highpressure Dionex ICS-5000+ HPIC system was used in this
Table 1. Six Stable Isotope-Labeled Internal Standards for the Current Study no.
metabolite name
formula
obsd m/z
ion
RT (min)
1
sodium pyruvate (13C3, 99%) succinic acid [13C4, 99%)] malic acid (13C4, 99%) alpha-Ketoglutaric acid (13C5, 99%) fumaric acid (13C4, 99%) citric acid (2,2,4,4-D4, 98%)
[13] C3H4O3 [13] C4H6O4 [13] C4H6O5 [13] C5H6O5 [13] C4H4O4 C6H4[2] H4O7
90.0188
[M − H]−
3.27
121.0328
[M − H]−
6.70
137.0277
[M − H]−
6.72
150.0310
[M − H]−
8.07
119.0172
[M − H]−
8.63
195.0449
[M − H]−
11.89
2 3 4 5 6
study mainly because a fast separation system with high chromatographic reproducibility is needed for targeted analysis of metabolites. The Dionex ICS-5000+ system can operate at a flow rate up to 380 μL/min with the pressure limit as high as 5000 psi (as compared to the previous application on the Dionex ICS 4000 HPIC capillary system at flow rate of 25 μL/ min9). High reproducibility for retention time (RT) and peak intensity can be achieved with the larger column capacity (2.0 mm, i.d.) over the capillary IC system (0.4 mm, i.d.). In addition, the transfer line from autosampler to injection valve was a red PEEK tubing (0.13 mm i.d.) with a 14 μL volume, which was found to substantially improve the sensitivity and peak shape as compared to a black (0.25 mm i.d.) or blue PEEK tubing (0.5 mm i.d.) with volumes greater than 40 μL. A C
DOI: 10.1021/acs.analchem.5b01350 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
Figure 2. (A) High-resolution IC with Q Exactive HF MS analysis of 11 sugar monophosphates and (B) 9 sugar diphosphates in UM1 cancer cells. Peak #9 eluting at 7.94 min was identified as fructose 6-phosphate based on matching MS/MS (insert) and retention time with the standard compound. The Q Exactive HF MS offers very accurate mass measurement of the parent and product ions at the negative mode (
