Mass Isotopomer Analysis of Nucleosides Isolated from RNA and DNA Using GC/MS

Santos, Ines Miranda; Gramacho, Silvia; Piñeiro, Marta; Martínez-Gómez, Karla; Fritz, Michel; Hollemeyer, Klaus; Salvador, Armindo; Heinzle, Elmar

doi:10.1021/ac503305w

Cited by 15 publications

(9 citation statements)

References 31 publications

(76 reference statements)

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“…2B). Previous methods focusing on biomass pools have relied on targeted digestion of nucleotides, proteins, and glycans individually [38–40]. Our method instead allows profiling of all three classes of biosynthetic intermediates simultaneously.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance

Badur

Zhang

Metallo

2015

Biotechnology Journal

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To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications.

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Section: Resultsmentioning

confidence: 99%

Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance

Badur

Zhang

Metallo

2015

Biotechnology Journal

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“…In later work, Miranda‐Santos et al () have used the derivatives and GC/MS to investigate metabolism with 13 C‐labeling.…”

Section: Trimethylsilyl Derivativesmentioning

confidence: 99%

Mass Spectrometric Fragmentation of Trimethylsilyl and Related Alkylsilyl Derivatives

Harvey

Vouros

2019

Mass Spectrometry Reviews

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This review describes the mass spectral fragmentation of trimethylsilyl (TMS) and related alkylsilyl derivatives used for preparing samples for analysis, mainly by combined gas chromatography and mass spectrometry (GC/MS). The review is divided into three sections. The first section is concerned with the TMS derivatives themselves and describes fragmentation of derivatized alcohols, thiols, amines, ketones, carboxylic acids and bifunctional compounds such as hydroxy‐ and amino‐acids, halo acids and hydroxy ethers. More complex compounds such as glycerides, sphingolipids, carbohydrates, organic phosphates, phosphonates, steroids, vitamin D, cannabinoids, and prostaglandins are discussed next. The second section describes intermolecular reactions of siliconium ions such as the TMS cation and the third section discusses other alkylsilyl derivatives. Among these latter compounds are di‐ and trialkyl‐silyl derivatives, various substituted‐alkyldimethylsilyl derivatives such as the tert‐butyldimethylsilyl ethers, cyclic silyl derivatives, alkoxysilyl derivatives, and 3‐pyridylmethyldimethylsilyl esters used for double bond location in fatty acid spectra. © 2019 Wiley Periodicals, Inc. Mass Spec Rev 0000:1–107, 2019

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“…However, several alternative measurement techniques are available, and in some cases, may be preferred: tandem mass spectrometry (Antoniewicz, 2013b; Choi and Antoniewicz, 2011; Choi et al, 2012), LC-MS/MS (McCloskey et al, 2016), and NMR (Masakapalli et al, 2014; Szyperski, 1995; Tang et al, 2007). Moreover, in addition to measuring labeling of protein-bound amino acids, it may be advantageous to measure 13 C-labeling of carbohydrates (McConnell and Antoniewicz, 2016), fatty acids (Crown et al, 2015b), nucleosides (Miranda-Santos et al, 2015), glycogen and RNA (Guzman et al, 2014; Long et al, 2016a), and intracellular metabolites (Ahn and Antoniewicz, 2013; Millard et al, 2014). Finally, a practical consideration is the cost associated with performing 13 C-MFA studies: the cost of tracers (Table 1), analytical instruments, sample preparation, analysis time, and the cost of performing parallel labeling experiments vs. single tracer experiments (Hollinshead et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system

Crown

Long

Antoniewicz

2016

Metabolic Engineering

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13C-Metabolic flux analysis (13C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13C-MFA. The best single tracers were doubly 13C-labeled glucose tracers, including [1,6-13C]glucose, [5,6-13C]glucose and [1,2-13C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6-13C]glucose and [1,2-13C]glucose. Combined analysis of [1,6-13C]glucose and [1,2-13C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1-13C]glucose + 20% [U-13C]glucose.

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Mass Isotopomer Analysis of Nucleosides Isolated from RNA and DNA Using GC/MS

Cited by 15 publications

References 31 publications

Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance

Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance

Mass Spectrometric Fragmentation of Trimethylsilyl and Related Alkylsilyl Derivatives

Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system

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