Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols

Abstract

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid-base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid-base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

“…Metabolite Extraction for LC-MS-The metabolites were extracted according to a procedure slightly modified from previous references (20,21). Basically, collected HeLa cells were added with 500 l of 80% methanol (20% water) solvent and lysed by a freeze-thaw cycle repeated three times.…”

Cell phenotypic changes of mouse connective tissue fibroblasts (L-929) to poly(ethylene glycol)-based gels

“…Metabolite extraction from cells was conducted as previously described. 24 Briefly, 400 μl of ice-cold extraction solvent, i.e. methanolchloroform-water (1 : 1 : 0.1, v/v/v), were added to the frozen cell pellet.…”

“…A mixture of chloroform, methanol and water is applicable for the extraction of intracellular metabolites, and was used to recover the maximum amount of metabolites. 24 As a result, both polar and lipophilic metabolites are extracted. GC-MS based metabolite profiling led to the identification of 15 analytes belonging to diverse biochemical classes such as amino acids (aspartic acid, glutamic acid, glycine and tyrosine), fatty acids (arachidic acid, myristic acid, palmitic acid, stearic acid), monosaccharides (fructose and glucose 6-phosphate), nucleotides (adenosine monophosphate (AMP)), polyamines ( putrescine), polyols (glucitol, myo-inositol), and steroids (cholesterol), Fig.…”

Hsp90 Inhibition Affects Cell Metabolism by Disrupting Mitochondrial Protein Insertion

“…After 24 h, cells were washed once with PBS and media containing 4 mM [U- 13 C] glucose was added. After 48h, cells were harvested and extracted as previously described [18] Labelled and unlabelled serine and glycine in 10 µL of media were derivatized with propyl chloroformate and analysed by HPLC-ESI-MS/MS as described previously [19]. The MRM transitions were programmed separately for unlabeled and fully labelled serine and glycine.…”