LC-MS/MS MRM Method and Raw Chromatography Data for Amino Acid Quantification in Tumor Cell Lysates and Conditioned Media

OverviewThis dataset contains the analytical method and the corresponding Agilent MassHunter analysis reports for the quantification of amino acids in tumor cell lysates and tumor cell culture media (TCM). The data was acquired using Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) in Multiple Reaction Monitoring (MRM) mode. Sample Preparation and Biological ContextAmino acid concentrations were analyzed in Hepa1-6BL-control and Hepa1-6BL-Slc1a4 KO cells. Cells (3 × 10^6) were cultured in 10 cm dishes with RPMI 1640 medium for 36 hours. Tumor cell culture supernatants (TCM) were collected, filtered (0.22 μm), and stored at -80°C. Cell lysates were prepared via repeated freeze-thaw cycles and filtration. Samples (20 µl) were mixed with an acetonitrile solution containing 0.1% formic acid, vortexed, and centrifuged (14,000 g, 4°C, 10 min) prior to analysis. LC-MS/MS Instrumentation and Method Instrumentation: Agilent 1290 UHPLC system coupled with an Agilent 6470B triple quadrupole mass spectrometer. Chromatography: Separation was achieved using an Agilent InfinityLab Poroshell 120 HILIC-Z column (2.1 mm × 150 mm, 2.7 μm). The mobile phase consisted of 10% (200 mM ammonium formate, pH=3) in ultrapure water (Phase A) and 10% (200 mM ammonium formate, pH=3) in acetonitrile (Phase B). A gradient elution was performed at 0.8 mL/min. The column temperature was maintained at 30°C, with a 5 μL injection volume. Mass Spectrometry: Positive electrospray ionization (ESI+) mode with MRM. Key parameters include: drying gas temp 300°C, drying gas flow 7 L/min, nebulizer pressure 45 psi, sheath gas temp 400°C, sheath gas flow 11 L/min, capillary voltage 3500 V. GABA-d6 was used as the internal standard. MRM Transitions: Specific transitions and collision energies were optimized for Cys, Thr, Ser, and Ala (detailed in the accompanying method file). SAMPLE IDENTIFICATION INFORMATIONS Sample name TCM (Sample ID) Tumor cell lysate (Sample ID) 1. sgControl P1-A4 P1-A10 2. sgControl P1-A5 P1-A11 3. sgControl P1-A6 P1-B1 1. sgSlc1a4 P1-A7 P1-B2 2. sgSlc1a4 P1-A8 P1-B3 3. SgSlc1a4 P1-A9 P1-B4