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Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies
Published on: January 23, 2019
Interlaboratory comparison for quantitative primary metabolite profiling in Pichia pastoris
Kristaps Klavins1, Stefan Neubauer, Ali Al Chalabi
1Austrian Centre of Industrial Biotechnology, Muthgasse 11, 1190 Vienna, Austria.
Analytical and Bioanalytical Chemistry
|April 23, 2013
Summary
This study compared metabolite profiling methods in Pichia pastoris, finding that isotope dilution upon extraction improved accuracy and reduced uncertainty in metabolite quantification. The interlaboratory comparison confirmed accurate quantification for most metabolites.
Area of Science:
- Metabolomics and Systems Biology
- Microbial Physiology and Fermentation Technology
- Analytical Chemistry and Mass Spectrometry
Background:
- Accurate quantitative metabolite profiling is crucial for understanding microbial metabolism, particularly in industrial strains like Pichia pastoris.
- Interlaboratory comparisons are essential for validating and harmonizing different analytical platforms and quantification strategies.
- Internal standardization is a key technique for improving the accuracy and reliability of quantitative metabolite measurements.
Purpose of the Study:
- To conduct the first interlaboratory comparison for quantitative metabolite profiling in Pichia pastoris.
- To evaluate different measurement platforms and internal standardization strategies for metabolite quantification.
- To assess the uncertainty contributions from measurement and sample preparation in metabolite profiling.
Main Methods:
- Two laboratories (BOKU and Biocrates) performed quantitative metabolite profiling on Pichia pastoris samples.
- Laboratory 1 used in vivo U-13C labeled internal standards upon extraction, coupled with RP-LC-MS/MS and HILIC-MS/MS.
- Laboratory 2 utilized a metabolomics kit with immobilized internal standards and automated sample preparation, coupled with HILIC-MS/MS and RP-LC-MS/MS.
Main Results:
- Intracellular metabolite concentrations ranged from 0.2 to 108 μmol g(-1) cell dry weight.
- Laboratory 1, using isotope dilution upon extraction, showed lower sample preparation uncertainty compared to Laboratory 2.
- Total combined uncertainties (K=2) were 21–48% for Laboratory 1 and 30–57% for Laboratory 2, with sample preparation being the major contributor.
Conclusions:
- The interlaboratory comparison was successful, with most metabolite concentrations agreeing within their uncertainties, indicating accurate quantification.
- Isotope dilution upon extraction is critical for accurate quantification, especially for redox-sensitive metabolites like methionine.
- The precision of the extraction procedure significantly impacts the overall uncertainty in quantitative metabolite profiling.
