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

Carnicer M, Aljoscha Wahl S, Seifar RM, Albiol J, van Gulik W, Ferrer P (2026)

Publication Type: Book chapter / Article in edited volumes

Publication year: 2026

Journal

Methods in Molecular Biology Humana Press

Publisher: Humana Press Inc.

Series: Methods in Molecular Biology

Book Volume: 2697

Pages Range: 137-152

DOI: 10.1007/978-1-0716-4779-0_8

Abstract

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.

Involved external institutions

Autonomous University of Barcelona (UAB) / Universitat Autònoma de Barcelona ES Spain (ES) Delft University of Technology (TU Delft) NL Netherlands (NL) Institut Químic de Sarrià (IQS) ES Spain (ES)

How to cite

APA:

Carnicer, M., Aljoscha Wahl, S., Seifar, R.M., Albiol, J., van Gulik, W., & Ferrer, P. (2026). ID-MS-Based Quantitative Analysis of Metabolites in Pichia pastoris: A Step-by-Step Protocol. In (pp. 137-152). Humana Press Inc..

MLA:

Carnicer, Marc, et al. "ID-MS-Based Quantitative Analysis of Metabolites in Pichia pastoris: A Step-by-Step Protocol." Humana Press Inc., 2026. 137-152.

BibTeX: Download