Compartment‐specific metabolomics for CHO reveals that ATP pools in mitochondria are much lower than in cytosol

Abstract

Mammalian cells show a compartmented metabolism. Getting access to subcellular metabolite pools is of high interest to understand the cells' metabolomic state. Therefore a protocol is developed and applied for monitoring compartment-specific metabolite and nucleotide pool sizes in Chinese hamster ovary (CHO) cells. The approach consists of a subtracting filtering method separating cytosolic components from physically intact mitochondrial compartments. The internal standards glucose-6-phosphate and cis-aconitate were chosen to quantify cytosolic secession and mitochondrial membrane integrity. Extracts of related fractions were studied by liquid chromatography-isotope dilution mass spectrometry for the absolute quantification of a subset of glycolytic and tricarboxylic acid cycle intermediates together with the adenylate nucleotides ATP, ADP and AMP. The application of the protocol revealed highly dynamic changes in the related pool sizes as a function of distinct cultivation periods of IgG1 producing CHO cells. Mitochondrial and cytosolic pool dynamics were in agreement with anticipated metabolite pools of independent studies. The analysis of adenosine phosphate levels unraveled significantly higher ATP levels in the cytosol leading to the hypothesis that mitochondria predominantly serve for fueling ATP into the cytosol where it is tightly controlled at physiological adenylate energy charges about 0.9.

Absolute Quantification of Matrix Metabolites Reveals the Dynamics of Mitochondrial Metabolism

“…Standard techniques for purifying mitochondria can take hours to complete, leading to the significant loss of metabolites because solute transporters and enzymes can have residual activity even at low temperatures (Bowsher and Tobin, 2001; Matuszczyk et al , 2015; Ross-Inta et al , 2008). Commercially available kits for antibody-based isolation of mitochondria are technically less cumbersome than other methods but utilize long immunopurification and wash steps (Miltenyi, 2011).…”

“…Commercially available kits for antibody-based isolation of mitochondria are technically less cumbersome than other methods but utilize long immunopurification and wash steps (Miltenyi, 2011). Abbreviated centrifugation protocols, selective membrane permeabilization, and non-aqueous fractionation all improve the speed of the workflow and have provided important insights into the metabolism of subcellular compartments, but the resulting mitochondrial preparations can be contaminated with cytosolic material, as well as other organelles, such as endoplasmic reticuli and lysosomes (Berry et al , 1991; Berthet and Baudhuin, 1967; Bestwick et al , 1982; Fly et al , 2015; Linskens et al , 2012; Matuszczyk et al , 2015; Roede et al , 2012; Tischler et al , 1977). Furthermore, components of traditional organellar isolation buffers (e.g., sucrose) can severely interfere with MS-based metabolite profiling (Roede et al , 2012).…”

Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

“…Standard methods for purifying mitochondria can take hours to complete and are thus too slow for profiling the metabolic contents of mitochondria. Existing methods that offer accelerated isolation procedures can suffer from poor purity, resulting in metabolic extracts that contain not only mitochondrial metabolites, but also cytosolic, lysosomal, and peroxisomal contents 12,14–21 . As such, interrogation of the metabolic contents of mitochondria requires a method that is both rapid and specific.…”

“…For example, isolation methods that can give relatively pure mitochondria, such as sucrose-gradient centrifugation 30,31 or the Miltenyi immunopurification kits, take notably longer to complete than our workflow, leading to greater distortion of the mitochondrial metabolite profile. Abbreviated forms of centrifugation, non-aqueous fractionation, and selective membrane permeabilization all isolate mitochondria with great speed, but the resulting material can be contaminated with whole cells and other organelles, such as lysosomes and peroxisomes 12,14–20 . Lysosomes in particular can distort the metabolite profile of mitochondrial preparations because they contain a notable amount of amino acids (data not shown).…”

Quantification of Microbial Phenotypes

“…This can be especially important when dealing with compartmentalized microorganisms such as baker’s yeast. Since it is experimentally challenging to extract metabolites from separate compartments independently, to the best of our knowledge, there is only one publication that has been able to measure compartment specific metabolites concentration, where cytosolic and mitochondrial CHO cells metabolites were quantified [21]. TMFA, which uses a similar approach, focuses on finding thermodynamically feasible flux distributions by exploiting the directional constraints on reactions for which the feasible Δ r G range is either strictly negative or strictly positive.…”