Glycolysis Pathway Analysis Service Case Study

Title: Comprehensive coverage of glycolysis and pentose phosphate metabolic pathways by isomer-selective accurate targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry assay

Journal: Analytical Chemistry

Published: 2024

Background

The accurate liquid chromatography-tandem mass spectrometry analysis of phosphorylated isomers from glycolysis and pentose phosphate pathways is a challenging analytical problem in metabolomics due to extraction problems from the biological matrix, adherence to stainless steel surfaces leading to tailing in LC, and incomplete separation of hexose and pentose phosphate isomers. There has been a lot of research on the analysis of sugar phosphates, and many incremental improvements have been made over time. However, each of these approaches has some limitations. The goal of this study was to present an improved robust HILIC-ESI-MS/MS method for accurate quantification of all metabolites of the glycolysis and pentose phosphate pathways, overcoming peak shape and retention time repeatability problems (ascribed to HILIC) and securing assay specificity for the isomers while also considering potential interferences from other pathways, such as mannose and lactose metabolism.

Methods

HILIC-ESI-MS/MS

Results

As shown in Figure 1, the four pentose phosphates are also separated. Ru5P and Xu5P were only partially separated, while Ru1P and Ri5P are fully baseline-separated. This separation should be suitable to allow at least an estimation of Ru5P and Xu5P, while the other two pentose phosphates can be determined without assay specificity compromises. Moreover, all the other target metabolites also show satisfactory peak shapes, and other critical isomeric peak pairs were fully resolved (2PG and 3PG, GA3P and DHAP). Pyr and Lac, which must be separated to avoid M + 2 isotopologue overlap, were fully baseline-resolved. The method also covers several bis-phosphates (F16P2 and 2,3PG), as well as PRPP with phosphate at the 5-position and a pyrophosphate group in position 1 of ribose. They exhibit symmetric peaks as well, and all target metabolites can be separated within 20 min.

Glycolysis Pathway Analysis Service Case Study Figure 1. Extracted ion chromatogram and isomer separation of HMP analytes found in the HeLa cell sample extract (3 × 10⁶ cells) with optimized method conditions.

Erastin was used to induce ferroptosis in HEK293 cells, an immortalized human embryonic kidney cell line. Erastin inhibits the cystine–glutamate antiporter system Xc, leading to cysteine deprivation and cellular deficiency to synthesize the antioxidant glutathione. It is accompanied by reactive oxygen species (ROS) accumulation due to mitochondrial dysfunction and causes an avalanche of metabolic alterations. Figure 2A shows the heat map of the glycolysis and PPP metabolites of a two-group comparison (erastin-induced ferroptosis vs control, i.e., untreated HEK293 cells). Cluster analysis provides a classification according to the two groups based on the glycolytic and PPP metabolite concentration levels. A volcano plot in Figure 2B generated by comparing metabolite levels of these two groups by univariate statistics reveals that the glycolytic metabolites F16P2, 2PG, 2,3BPG, and 3PG were significantly upregulated (p-value of <0.05) with a fold-change of >2 in the erastin-induced ferroptosis group. Enhanced glycolysis in cells with ferroptosis was further confirmed by the enhanced glucose uptake.

Glycolysis Pathway Analysis Service Case Study Figure 2. (A) Heatmaps displaying an overview of metabolite abundance distribution, with comparison between erastin-induced ferroptotic cell samples (ER) versus the control (CTRL). (B) Volcano plots showing the comparison between ferroptosis-induced (erastin) samples and the control (p-value threshold 0.05). (C) Representative boxplots of significantly increased metabolites (2PG, 3PG, 2,3BPG, and F16P2) found in HEK293 cell extract samples in which ferroptosis was induced compared to untreated ones (control).^[1]

Reference

Serafimov K.; et al. Comprehensive coverage of glycolysis and pentose phosphate metabolic pathways by isomer-selective accurate targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry assay. Anal Chem. 96(43):17271-17279. https://pubs.acs.org/doi/full/10.1021/acs.analchem.4c03490

* For Research Use Only. Not for use in diagnostic procedures.

Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

Great Minds Choose Creative Proteomics