Dataset Information

Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.

ABSTRACT:

Introduction

Accurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography-mass spectrometry are predominantly used to target polar metabolites.

Objectives

This study aims to demonstrate a simple one-step extraction combined with liquid chromatography-mass spectrometry (LC-MS) that reliably retains polar metabolites.

Methods

The method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography-mass spectrometry (LC-MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as - 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method's consistency and recovery efficiency.

Results

The method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of - 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70-130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson's correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample.

Conclusion

This LC-MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.

SUBMITTER: Day F

PROVIDER: S-EPMC11252196 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Publications

Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.

Day Francesca F O'Sullivan Justin J Ramzan Farha F Pook Chris C

Metabolomics : Official journal of the Metabolomic Society 20240716 4

<h4>Introduction</h4>Accurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography-mass spectrometry are predominantly used to target polar metabolites.<h4>Objectives</h4>This study aims to demonstrate a simple one-step extraction combined with liquid chromatography-mass spectrometry (LC-MS) that reliably retains polar metabolites.<h4>Method ...[more]

PMID: 39014104

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Project description:Continual developments in instrumental and analytical techniques have aided in establishing rigorous connections between protein glycosylation and human illness. These illnesses, such as various forms of cancer, are often associated with poor prognoses, prompting the need for more comprehensive characterization of the glycoproteome. While innovative instrumental and computational strategies have largely benefited glycoproteomic analyses, less attention is given to benefits gained through alternative, optimized chromatographic techniques. Porous graphitic carbon (PGC) chromatography has gained considerable interest in glycomics research due to its mobile phase flexibility, increased retention of polar analytes, and improved structural elucidation at higher temperatures. PGC has yet to be systematically compared against or in tandem with standard reversed phase liquid chromatography (RPLC) in high-throughput bottom-up glycoproteomic experiments, leaving the potential benefits unexplored. Performing comparative analysis of single and biphasic separation regimes at a range of column temperatures illustrates complementary advantages for each method. PGC separation is shown to selectively retain shorter, more hydrophilic glycopeptide species, imparting higher average charge, and exhibiting greater microheterogeneity coverage for identified glycosites. Additionally, we demonstrate that liquid-phase separation of glycopeptide isomers may be achieved through both single and biphasic PGC separations, providing a means towards facile, multidimensional glycopeptide characterization. Beyond this, we demonstrate how utilization of multiple separation regimes and column temperatures can aid in profiling the glycoproteome in tumorigenic and aggressive prostate cancer cells. RAW MS proteomic and glycoproteomic datasets have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024196 (10.6019/PXD024196) and PXD024195, respectively.

Dataset Information

Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.

Introduction

Objectives

Methods

Results

Conclusion

Publications

Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase.

Similar Datasets

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