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  • Phosphatase Inhibitor Cocktail II br While numerous studies


    While numerous studies have examined the flux of carbon from glucose using mass spectrometry methods, this is the first to specifically examine incorporation into one carbon metabolism, specifically me-thionine. Due to the essential role of methionine and one carbon me-tabolism in establishing the epigenetic landscape and synthesis of nu-cleic acids, the approach presented here should prove valuable in understanding how metabolic changes can result in epigenetic repro-gramming and DNA replication.
    2. Materials and methods
    2.1. Chemicals and reagents
    Fig. 2. The Phosphatase Inhibitor Cocktail II GC/MS ion chromato-grams and mass spectra of internal standard amino acids.
    A. Selective ion monitoring (SIM) using the precursor (M+.)-57 ions. Show is the total-ion-current (TIC) elution profile of internal standard stable-isotope labeled (IS) amino acids Gly+5, Met+3, and Ser+7. The che-mical structures of the three IS Phosphatase Inhibitor Cocktail II are presented below the TIC. Red colored atoms are the stable-isotope labeled atoms.
    B. The full-scan spectra of IS amino acids including Gly+5 (top panel), Met+3 (middle panel) and Ser+7 (bottom panel). While molecular ions are not present, major fragmented ions are M+.-15, M+.-57, M+.-85, and M+.-159. In this study, M+.-57 ions were used for quantification using the SIM mode. (For interpretation of the refer-ences to color in this figure legend, the reader is referred to the Web version of this article.)
    2.2. Cell lines and cell culture conditions
    The cell lines, U251 and U87 were generously obtained from MD Anderson cancer center as previously described and A375 were ob-tained from ATCC [13]. The PHDGH over expressing cell line of A375 was generated as previously described [14]. DMEM media without glycine and serine containing glucose was prepared by adding 3.5 g of isotope labeled glucose (Gluc+13) to 1 L of DMEM media. Cells were grown in regular DMEM and 10% FBS and
    seeded into 6 well plates. Media was removed, cells were washed with PBS, and DMEM with Gluc+13, no serine, no glycine, and 10% FBS was
    added. Cells were grown under standard culture conditions until 80–90% confluence (~3 days).
    2.3. Extraction of amino acids and analysis by gas chromatography/mass spectrometry
    After removal of the media, cells were rinsed with cold PBS at 4° C twice before adding 700 μL 90% methanol at 4° C to lyse the cells. The lysate containing the cell contents was placed into a 2 mL Eppendorf 
    tube and sonicated on ice at 30 amps for 3 × 30 s for extraction of small molecules. The cell lysate was centrifuged for 5 min at 10,000 G. The pellet was used to estimate total protein content (1 mg protein = 100 μL cells). Filtered lysates (100 μL) were placed into GC-MS vials and 20 μL of an internal standard solution was added. The standard solution contained Ser+7, Gly+5 and Met+3 at a concentration of 2 × 10−5 M. The supernatant was filtered through a centrifugal filter to remove molecules larger than 3 kD.
    Samples were evaporated under reduced pressure. Acetonitrile
    (20 μL) and 20 μL of a solution containing MTBSTFA plus 1% TBDMCS were added, vials were sealed and then heated at 60 °C for 1 h. Three technical replicates and at least three biological replicates were pre-pared for each sample.
    2.4. Western blotting
    Relative expression of PHGDH and SHMT2 were measured by Western blot. Cell lysates containing ~30 μg of total protein per sample were electrophoresed on an SDS-PAGE gel and transferred to a ni-trocellulose membrane. Membranes were first probed with either anti-PHGDH or anti-SHMT2 and then washed and a second fluorescent an-tibody was added. Fluorescence was measured with a LI-COR Odyssey imaging system (Lincoln, NE, USA).
    2.5. Statistical analysis
    Determination of statistical significance of measurements among different treatment groups was completed using two-way ANOVA in GraphPad Prism v6 ( ). A web based Java code based on the Sattherwaite equation was written for calculation of the degrees of freedom (DF) and t-values that were used for calculation of p values [15]. Significance is defined as a p-value less than 0.05.
    3. Results
    3.1. Gas chromatography mass spectrometry isotope tracing
    Chromatographic separation of the TBDMS derivatives of glycine, serine and methionine is shown in Fig. 2A. The structures and formulas of the isotopomers of glucose, glycine, serine and methionine as well as the mass spectra of the stable isotope enriched internal standards are also shown (Fig. 2B). These data establish the identity and purity of the enriched amino acid isotopomers used here. Standard curves were constructed to establish the relationship be-tween amount of each amino acid and the integrated area of the se-lected ion peak and to demonstrate that peak size and concentration are linear (Table 1). In order to determine the method's ability to recover the measured amino acids we prepared a mixture of serine, glycine, and methionine at a defined concentration and spiked them in U87 cell ly-sates after the centrifugation step. This is the step where our isotope standards are also added. We followed our extraction protocol and measured the recovery rates after subtracting a blank sample where no prepared mixture was added (Table 2). Samples after derivatization were stable for at least two weeks. Repeated analyses had inter- and