Comprehensive Vitamer Profiling of Folate Mono- and Polyglutamates in Baker's Yeast (Saccharomyces cerevisiae) as a Function of Different Sample Preparation Procedures.

Lena Gmelch,Lisa Striegel,Michael Witting,Philippe Schmitt-Kopplin,Michael Rychlik,Nadine Weber,Daniela Wirtz

doi:10.3390/metabo10080301

Lena Gmelch, Lisa Striegel + Show 5 more

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https://doi.org/10.3390/metabo10080301

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Abstract

Folates are a group of B9 vitamins playing an important role in many metabolic processes such as methylation reactions, nucleotide synthesis or oxidation and reduction processes. However, humans are not able to synthesize folates de novo and thus rely on external sources thereof. Baker’s yeast (Saccharomyces cerevisiae) has been shown to produce high amounts of this vitamin but extensive identification of its folate metabolism is still lacking. Therefore, we optimized and compared different sample preparation and purification procedures applying solid phase extraction (SPE). Strong anion exchange (SAX), C18 and hydrophilic–lipophilic-balanced (HLB) materials were tested for their applicability in future metabolomics studies. SAX turned out to be the preferred material for the quantitative purification of folates. Qualification of several folate vitamers was achieved by ultra-high pressure liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-Q-ToF-MS) measurements and quantification was performed by liquid chromatography tandem mass spectrometry (LC-MS/MS) applying stable isotope dilution assays (SIDAs). The oxidation product s-pyrazino-triazine (MeFox) was included into the SIDA method for total folate determination and validation. Applying the best protocol (SAX) in regard to folate recovery, we analyzed 32 different vitamers in different polyglutamate states up to nonaglutamates, of which we could further identify 26 vitamers based on tandem-MS (MS2) spectra. Total folate quantification revealed differences in formyl folate contents depending on the cartridge chemistry used for purification. These are supposedly a result of interconversion reactions occurring during sample preparation due to variation in pH adjustments for the different purification protocols. The occurrence of interconversion and oxidation reactions should be taken into consideration in sample preparation procedures for metabolomics analyses with a focus on folates.

Highlights

Metabolomics studies usually are performed to obtain a deeper insight into the metabolism of the organism investigated [1,2,3]
To unravel differences in the applied purification procedures in more depth, we focused on the qualitative and quantitative analysis of metabolites belonging to the folate group
To study folate metabolism in yeast samples by UHPLC-Q-ToF-mass spectrometry (MS) measurements, we first tried to apply simple shotgun approaches, which usually are used in studies of the metabolite profile of yeasts [14,15,86]

Summary

Introduction

Metabolomics studies usually are performed to obtain a deeper insight into the metabolism of the organism investigated [1,2,3]. This information is used to elucidate metabolic pathways [4,5], or to determine biomarkers for diseases [6,7,8], or influences of outer circumstances on metabolism [9,10,11,12,13]. To avoid artifacts generated by degradation reactions, typical sample preparation procedures are Metabolites 2020, 10, 301; doi:10.3390/metabo10080301 www.mdpi.com/journal/metabolites. To avoid artifacts generated by degradation reactions, typical sample preparation procedures are rather gentle [3,14,15,16]. Mostly metabolites with the highest abundance are typically denaturing enzymes date, mostly metabolites with highest abundance areHowever, typically investigated [3,18,19],[1,3,17,18]

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