br intra day variation less than br
intra-day variation less than 10%.
As shown in Fig. 3, Gluc+13 is converted to Ser+4/Ser+5, Gly+2, and Met+2. The measured concentrations of these amino ML210 iso-topomers were calculated based on their relative peak size compared to the internal isotope standard, after correcting for the natural isotopic envelope (Fig. 4, Supplemental_calculations).
3.2. Comparing cell lines with differential PHGDH expression
To test the argument that PHGDH is the rate-limiting enzyme in de novo serine synthesis, three cell lines were chosen with varying levels of PHGDH expression. U251 had the least PHGDH expression while the cell line, A375, and a A375 cell line that overexpresses PHGDH had progressively increasing amounts. The derived cell line, A375 + PHGDH, expressed PHGDH at approximately 4 times the level of the parent cell line (Fig. 5A). While PHGDH expression varied, SHMT2 levels appeared to be equal across cell lines.
Flux through the pathway was consistent with protein expression by western blots as the contribution of glucose-derived serine increased with PHGDH expression (Fig. 6A). Interestingly, Ser+0 was also posi-tively correlated which suggests this pathway makes use of both de novo
Standard curves established for the analysis of amino acids by GC/MS.
Standard curve equation: y = fx + b
AA Internal f (slope) b (y-intercept) r2(regression)
Recovery rates for quantification of AAs.
Amino Acids (femtomoles on column) Recovery rate (%)
and extracellular serine for production of one-carbon units (Fig. 6C). Unlabeled serine and glycine come from the 10% FBS that was added as per standard cell culture conditions of cancer cells. However, DMEM was prepared without serine and glycine. Concentrations of serine and glycine in 10% FBS are expected to be ~30μM and ~60μM, respectively . Although isotopically labeled serine and glycine levels correlated with PHGDH expression, Met+2 levels remained unchanged across all cell lines and absolute Met+2 concentration is ~50-fold lower than either Ser+4/Ser+5 or Gly+2 levels (Fig. 5B,C,D). Further, relative to the total intracellular concentration of methionine, this pathway con-tributed only 1–2% of the total intracellular methionine pool (Fig. 6B). However, typical cell culture conditions contain 200 μM methionine. It was then somewhat surprising to have discovered that intracellular methionine concentrations were found to be significantly lower than that of DMEM, 4-10μM depending on the cell line (Fig. 6D).
3.3. A375 PHGDH overexpressing cell line treated with PHGDH and SHMT2 inhibitors
Experiments were then repeated with an inhibitor of PHGDH (PHGDHi) and SHMT2 (SHMT2i). Results are shown in Fig. 7 A-F. As anticipated, inhibition of PHGDH resulted in a concentration-depen-dent decrease in the conversion of Gluc+13 to labeled serine, glycine, and methionine while inhibition of SHMT2 also decreased glycine and methionine but expectedly increased the serine concentration.
We also wanted to see how the effects of these inhibitors would affect the unlabeled amino acid pool as this presumably represents flux of amino acids into the cell. For U251 cells treated with varying con-centrations of either PHGDHi or SHMT2i, neither Ser+0 nor Met+0 levels appeared to have changed (Fig. 8). In contrast, Ser+0 levels de-creased in A375 cells with PHGDHi but increased with SHMT2i which is consistent with the trend seen in labeled serine. However, Met+0 levels did not change despite Met+2 levels having been decreased with either inhibitor. In A375 PHGDH overexpressing cells, Ser+0 levels increased with 0.5 μM PHGDH inhibitor but then decreased at 10 μM inhibitor relative to cells not treated with any drug. Otherwise, trends in A375 + PHGDH were the same as seen in A375. The results here suggest that the different inhibitors may have cell-type dependent ef-fects on amino acid flux into cells.
We present here a method to monitor the de novo synthesis of serine, glycine, and methionine in human cancer cell lines as well as unlabeled intracellular amino acid pools. The metabolites are efficiently extracted from cultured cells, and they are easily separated and unambiguously identified and quantified by GC-MS. Good extraction is reproducibility obtained, and reliable quantitation is made when using isotope-
Fig. 3. Scheme for isotope tracking of glucose into
serine, glycine, and methionine.
3-phosphoglycerate (3-PG), formed at sixth step of gly-
colysis, by phosphoglycerate kinase (PGK) can be con-
verted into phosphohydroxypyruvate by phosphoglyce-