Selective and Accurate Quantification of N-Acetylglucosamine in Biotechnological Cell Samples via GC–MS/MS and GC–TOFMS

Abstract

N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. It plays a variety of roles at the cell surface structure and is under discussion to be involved in signaling pathways. The presence of a number of N-acetylhexosamine stereoisomers in samples of biological or biotechnological origin demands for dedicated high efficiency separation methods, due to identical exact mass and similar fragmentation patterns of the stereoisomers. Gas chromatography offers high sample capacity, separation efficiency, and precision under repeatability conditions of measurement, which is a necessity for the analysis of low abundant stereoisomers in biological samples. Automated online derivatization facilitates to overcome the main obstacle for the use of gas chromatography in metabolomics, namely, the derivatization of polar metabolites prior to analysis. Using alkoximation and subsequent trimethylsilylation, carbohydrates and their derivatives are known to show several derivatives, since derivatization is incomplete as well as highly matrix dependent inherent to the high number of functional groups present in carbohydrates. A method based on efficient separation of ethoximated and trimethylsilylated N-acetylglucosamines was developed. Accurate absolute quantification is enabled using biologically derived 13C labeled internal standards eliminating systematic errors related to sample pretreatment and analysis. Due to the lack of certified reference materials, a methodological comparison between tandem and time-of-flight mass spectrometric instrumentation was performed for mass spectrometric assessment of trueness. Both methods showed limits of detection in the lower femtomol range. The methods were applied to biological samples of Penicillium chrysogenum cultivations with different matrices revealing excellent agreement of both mass spectrometric techniques.

Benchmarking Non-Targeted Metabolomics Using Yeast-Derived Libraries

“…Literature search confirmed our findings and additionally expanded the reported compounds [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ] in our library, which can be found in the Supplementary Table S1 (Excel sheet FinalMetaboliteList). As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and humans [ 19 ].…”

“…Over the recent past, yeast-based standards have seen a slow but increasing acceptance in the metabolomics community. Several laboratories resorted to Pichia pastoris ethanolic yeast extracts [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ] primarily for 13 C internal standardization. Only a few studies were related to instrument performance tests or method development in accordance with the here proposed application [ 30 , 35 , 36 , 37 ].…”

“…As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and humans [ 19 ]. The curated list includes 206 metabolites and lipids (expected, detected, quantified in HMDB) present in the ethanolic yeast extract using orthogonal RP-LC-HRMS and HILIC-HRMS workflows, of which 181 metabolites were reproducibly identified (at least in two different fermentation batches, different instruments, two participating laboratories or others [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]). The additional 25 metabolites were reported elsewhere [ 27 , 28 , 29 , 30 , 33 , 34 ] ( Supplementary Table S1 , column, FinalMetaboliteList, “Additional literature hits”).…”

Benchmarking non-targeted metabolomics using yeast derived libraries

“…The yeast extract was produced from controlled Pichia pastoris (Guillierm.) Phaff 1956 (Komagataella phaffii Kurtzman) 27 Literature search confirmed our findings and additionally expanded the reported compounds [30][31][32][33][34][35][36][37][38][39] in our library which can be found in the Supplementary two participating laboratories or others [30][31][32][33][34][35][36][37][38][39] ). The remaining 25 metabolites were reported elsewhere [30][31][32][33]36,37 ( Supplementary Table S1, column, FinalMetaboliteList, "Additional literature hits").…”

“…Over the recent past, yeast-based standards have seen a slow but increasing acceptance in the metabolomics community. Several laboratories resorted to Pichia pastoris ethanolic yeast extracts [30][31][32][33][34][35][36][37][38][39] primarily for 13 C internal standardization. Only a few studies were related to instrument performance tests or method development in accordance to the here proposed application 33,[38][39][40] .…”