ID-MS-Based Quantitative Analysis of Metabolites in Pichia pastoris: A Step-by-Step Protocol.

Quantitative metabolomics is based on a set of experimental approaches to accurately quantify intracellular metabolite concentrations. This allows us to characterize the response of a metabolic network (i.e., the metabolic phenotype) to an environmental or genetic perturbation. Here, we describe a four-step protocol adapted to the methylotrophic yeast Komagataella phaffii: (1) separation of the cells from the fermentation broth by cold filtration and addition of 13C-labeled cell extract, (2) a metabolic quenching step based on aqueous cold methanol, (3) a metabolite extraction method based on boiling ethanol, and (4) quantification by isotope dilution mass spectrometry (LC-IDMS/MS and/or GC-IDMS). This method allows us to quantify most metabolites of central carbon metabolism, including glycolytic, tricarboxylic acid cycle, and pentose phosphate pathway intermediates, as well as cofactors and free amino acids. This method has been validated for K. phaffii grown on glucose, as well as on a mixture of carbon substrates such as methanol in combination with glucose or glycerol.