br Although potential applications of metabolomics findings
Although potential applications of metabolomics findings until now are encouraging and their significance is doubtless, limited work has focused on targeted plasma metabolic profiling of BC for accurate di-agnosis and pathogenesis clarification. Few metabolomics studies of BC biomarker discovery have, to date, focused on multicenter replication and validation. In this study, a targeted plasma metabolic profiling approach optimized for the detection of over 400 metabolites reflective of > 35 metabolic pathways of potential biological relevance is re-ported. A total of 201 plasma samples from BC and control subjects from two different clinical centers were analyzed, and potential bio-markers were selected by means of univariate significance testing and multivariate model construction and validation. In the current study, two sets of control samples taken from two collection sites were ana-lyzed, and only metabolites that were not found to be significantly Journal of Chromatography B 1105 (2019) 26–37
different between control groups were retained for further comparison of metabolic profiles between breast cancer patients and their healthy counterparts (see Supplemental Scheme S1).
2. Material and methods
Acetonitrile (ACN), methanol (MeOH), acetic 2070009-61-7 (AcOH), and ammonium acetate, all LC-MS grade, were purchased from Fisher Scientific (Pittsburgh, PA). Standard compounds (purity > 95–99%) corresponding to measured metabolites were purchased from Sigma-Aldrich (Saint Louis, MO) and Fisher Scientific (Pittsburgh, PA). Internal standards (stable 13C-labeled tyrosine and lactate; purity > 99%) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA).
2.2. Clinical samples
Clinical samples were purchased from the Fred Hutchinson Cancer Research Center Breast Specimen Repository (FHCRC; Seattle, WA) and Bloodworks Northwest (Seattle, WA). This analysis was deemed IRB exempt, since these samples were commercially purchased. Informed consent was obtained from all participants (both BC patients and healthy controls) before sample collection at the aforementioned re-search institutes. All participants were evaluated, and blood samples were obtained after overnight fasting. In total, 201 subject samples were included in the study, among which there were 102 BC patients and 99 healthy controls. The controls were age-matched with BC pa-tients (see Table 1 for full clinical and demographic characteristics of study participants). Among the controls, 31 of 99 samples were allo-cated from FHCRC (Seattle, WA), while 68 control samples were allo-cated from Bloodworks Northwest (Seattle, WA). All clinical samples of BC subjects were allocated from FHCRC.
2.3. Sample preparation
The sample preparation protocol was modeled on previous studies [37,38]. Frozen samples were thawed overnight under 4 °C, and 50 μL
Demographic and clinical characteristics of study participants.
Breast cancer Healthy controls
Perimenopausal 6 (5.9)
of each plasma sample was placed in a 2 mL Eppendorf vial. Protein precipitation and metabolite extraction were performed by adding 300 μL of methanol. The mixture was then vortexed for 2 min and stored at −20 °C for 30 min, followed by sonication in an ice bath for 10 min and subsequent centrifugation at 14,000 RPM for 20 min at 4 °C. The supernatant (150 μL) was collected into a new Eppendorf vial, and dried using a Vacufuge Plus evaporator. The dried samples were re-constituted in 500 μL of 5 mM ammonium acetate in 40% H2O/60% ACN + 0.2% acetic acid containing 5.13 μM L-tyrosine-13 C2 and 22.5 μM sodium-L-lactate-13C2. The two stable isotope-labeled internal standards were added to each sample to monitor system performance. A pooled sample, which was a mixture of plasma from all BC patients and healthy controls, was extracted using the same procedure as previously described. This sample was used for quality control (QC) purposes and was analyzed once every 10 study samples.
2.4. Liquid chromatography and mass spectrometry conditions
The targeted LC-MS/MS method used here was modeled after that developed and used in a growing number of studies [20,30,39–43]. Briefly, LC-MS/MS experiments were performed on a Waters Acquity I-Class UPLC TQS-micro MS system (Milford, MA). Each sample was in-jected twice, 2 μL and 5 μL for analysis using positive and negative io-nization modes, respectively. Chromatographic separation was per-formed on a Waters Xbridge BEH Amide column (2.5 μm, 2.1 × 150 mm) at 40 °C. The flow rate was 0.3 mL/min. For positive mode, the mobile phase was composed of Solvents A (5 mM ammonium acetate in H2O with 0.1% acetic acid) and B (ACN with 0.1% acetic acid). For negative mode, Solvent A was 10 mM ammonium bicarbo-nate in H2O, and Solvent B was ACN. The LC gradient conditions were the same for both positive and negative ionization modes. After an initial 1.5 min isocratic elution of 10% Solvent A, the percentage of Solvent A was increased linearly to 65% at t = 9 min. The percentage of A then remained the same (65%) for 5 min (t = 14 min), after which the percentage of A was reduced to 10% at t = 15 min to prepare for the next injection. The total experimental time for each injection was 30 min. Metabolite identities were confirmed by spiking mixtures of standard compounds into prepared plasma samples. Extracted MRM peaks were integrated using the TargetLynx software (Waters, Milford, MA).