Glycolysis Metabolomics Service - Quantify Glycolytic Pathways

Q: How does your service distinguish between G6P and F6P isomers?

Glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) are isobaric, so mass spectrometry alone cannot separate them. We use optimized hydrophilic interaction liquid chromatography (HILIC) to achieve baseline chromatographic separation before MS detection. This ensures independent, accurate quantification of G6P and F6P, rather than a merged signal.

Q: Why is LC–MS/MS superior to colorimetric or fluorometric assay kits for glycolysis analysis?

Assay kits are useful for single readouts (e.g., lactate) but are limited in specificity, multiplexing, and pathway coverage. Our LC–MS/MS platform simultaneously quantifies multiple glycolytic intermediates (e.g., G6P, F1,6BP, PEP, pyruvate, lactate) and energy cofactors (ATP/ADP), enabling pathway-level interpretation, flux ratios, and identification of enzymatic bottlenecks that single-analyte kits cannot provide.

Q: Can this platform quantify energy charge (ATP/ADP/AMP) and redox state (NAD⁺/NADH)?

Yes. Our targeted panels can include ATP, ADP, AMP, NAD⁺, and NADH. We use dedicated extraction protocols to preserve these labile cofactors, allowing you to directly link glycolytic changes with cellular energy charge and redox status.

Q: Why is "metabolic quenching" required for glycolysis samples?

Glycolytic intermediates and energy carriers turn over within seconds. Without rapid quenching, ongoing enzymatic activity distorts metabolite levels. We provide SOPs for immediate quenching (e.g., liquid nitrogen or cold organic solvents) to “freeze” the metabolic state at the time of sampling

Q: What are the minimum sample requirements for rare cell populations (e.g., FACS-sorted cells)?

For standard workflows we recommend ~1×10⁶ cells per sample, but using our low-input protocols we can typically work with 50,000–100,000 cells (e.g., specific T-cell subsets, stem cells). Please discuss available cell numbers with us during the consultation so we can adjust extraction volumes accordingly.

Q: Can I use formalin-fixed paraffin-embedded (FFPE) tissues for this service?

No. FFPE processing removes most polar metabolites and damages labile intermediates, making it unsuitable for glycolysis-focused metabolomics. We require fresh-frozen tissues, snap-frozen in liquid nitrogen and stored at −80 °C.

Q: Is the data provided as absolute concentration or relative abundance?

By default, we provide normalized peak areas for robust group comparisons. Absolute quantification can be offered for selected metabolites using multi-point calibration and stable isotope–labeled internal standards.

Q: How do you handle the high variability often seen in glucose and lactate levels?

We reduce variability using isotopically labeled internal standards, strict QC, and drift correction. In addition to glucose and lactate, we recommend focusing on intracellular intermediates (e.g., F1,6BP, PEP, 6-PG), which provide a more stable and informative readout of glycolytic and PPP flux.

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Why Quantify Glycolysis in Your Research?

Glycolysis is a central pathway that supports:

Tumor growth and metabolic reprogramming
Immune cell activation and effector functions
Obesity, diabetes, and other metabolic disease models
Microbial fermentation and strain engineering
Drug mechanism of action and toxicity studies

Because many glycolytic intermediates are polar, labile, and low-abundance, they are difficult to measure with generic assays. A dedicated, targeted metabolomics platform is needed to move beyond simple glucose or lactate readouts and obtain a pathway-level view of glycolysis.

Glycolysis Targeted Metabolomics Service Overview

Our Glycolysis Targeted Metabolomics Service provides LC–MS/MS–based quantification of key glycolytic intermediates, with optional coverage of related central carbon metabolism nodes (e.g., TCA intermediates).

With this service, you can:

Quantify glycolytic intermediates across cells, tissues, body fluids, and microbial samples
Compare metabolic states between control vs. treated, wild-type vs. knockout, responder vs. non-responder
Assess how drugs, gene perturbations, diet, or process changes reshape glycolytic flux

Glycolysis Targeted Metabolite Panels

We provide several glycolysis-focused targeted metabolite panels that can be used alone or combined, depending on your study design and pathway coverage needs.

Core Glycolysis Metabolite Panel (Standard)

This LC–MS/MS panel is designed for pathway-level profiling of glycolysis and includes isomer-resolved measurement of key intermediates.

Segment	Metabolites (examples)
Upper glycolysis	Glucose-6-phosphate (G6P); Fructose-6-phosphate (F6P); Fructose-1,6-bisphosphate (F1,6BP)
Mid-pathway	Glyceraldehyde-3-phosphate (G3P); Dihydroxyacetone phosphate (DHAP); 3-phosphoglycerate (3-PG); 2-phosphoglycerate (2-PG)
Late glycolysis/output	Phosphoenolpyruvate (PEP); Pyruvate; Lactate

* G6P and F6P are chromatographically separated to distinguish glycolysis from PPP-related flux.

Extended Panels for Central Carbon Metabolism

For projects that require a broader view of central carbon metabolism, you can add one or more of the following targeted panels:

Panel	Typical Metabolites (examples)	Typical Use Cases
Glycolysis + Energy Cofactor Panel	ATP, ADP, AMP, NAD⁺, NADH (measured together with glycolytic intermediates)	Linking glycolytic flux to cellular energy charge and redox balance
Glycolysis + TCA Cycle Panel	Citrate, cis-aconitate, α-ketoglutarate, succinate, fumarate, malate	Coordinated analysis of glycolysis and mitochondrial TCA activity
Glycolysis + PPP Panel	6-phosphogluconate (6-PG), ribose-5-phosphate (R5P), sedoheptulose-7-phosphate	Investigating NADPH production, oxidative stress, nucleotide synthesis
Glycolysis + Hexosamine Pathway Panel	Glucosamine-6-phosphate (GlcN6P), UDP-N-acetylglucosamine (UDP-GlcNAc)	Studying protein glycosylation, signaling, and nutrient sensing

Panels can be combined or customized, enabling you to build a glycolysis-focused targeted metabolomics panel that matches your specific pathway and flux questions.

Targeted vs. Untargeted Metabolomics for Glycolysis

Aspect	Untargeted Metabolomics	Targeted Glycolysis Metabolomics
Purpose	Discovery-driven; Global metabolic profiling	Quantitative, pathway-level analysis of glycolysis
Best For	Identifying unknown metabolic changes, early-stage studies	Validating metabolic hypotheses, mechanism-of-action studies
Metabolite Coverage	Broad, covering many metabolic pathways	Focused on key glycolytic intermediates and related pathways
Glycolytic Metabolite Resolution	Limited; possible poor resolution of glycolytic isomers (e.g., G6P/F6P)	High-resolution; G6P/F6P separation for precise pathway flux
Energy Co-Factors	Not typically measured in untargeted workflows	ATP, ADP, AMP, NAD⁺, NADH can be included alongside glycolytic intermediates
Pathway Depth	Limited; may miss key branches like PPP, HBP	Comprehensive, covering glycolysis, PPP, TCA, and more
Data Confidence	Lower reproducibility for specific metabolites	High reproducibility and quantification
Typical Use Case	Exploration phase: Identifying broad metabolic shifts	Validation phase: Targeted analysis of glycolysis and energy metabolism

Recommended Approach

For discovery-focused studies, you can use untargeted metabolomics to identify key metabolic changes across pathways, and then use targeted glycolysis metabolomics to validate and quantify specific intermediates of interest. This two-step approach provides both global insights and detailed pathway analysis.

Key Advantages of Our Glycolysis Metabolism Service

Accurate Quantification of Unstable Metabolites
Optimized LC–MS/MS methods ensure reliable detection of polar, low-abundance glycolytic intermediates.
Isomer Separation of G6P and F6P
We resolve key isomers like glucose-6-phosphate and fructose-6-phosphate, enabling clear analysis of glycolysis vs. PPP flux.
Integrated Detection of Energy Cofactors
ATP, ADP, AMP, NAD⁺, and NADH can be analyzed alongside glycolytic metabolites in a single workflow.
Coverage of Glycolysis-Linked Pathways
Optional inclusion of pentose phosphate pathway metabolites (e.g., 6-PG, R5P) and other central carbon nodes.
Support for Low-Input and Precious Samples
Extraction protocols support small tissue samples and limited cell numbers, including sorted immune cell subsets.
Validated Rapid Quenching & Sample Handling SOPs
We offer expert guidance on rapid quenching to preserve labile intermediates in cells and tissues.
Pathway-Level Biological Insight
Results include statistical analysis, visualizations, and interpretation support to reveal metabolic rewiring.

LC–MS/MS Platform for Glycolysis Metabolite Quantification

Instrumentation

LC–MS/MS platform: Triple quadrupole mass spectrometer

Separation methods:

HILIC (hydrophilic interaction chromatography) for polar metabolites
Reverse-phase LC for energy cofactors and hydrophobic metabolites

Detection mode: Multiple Reaction Monitoring (MRM/SRM)

Quantification & QC

Item	Details
Quantification method	Internal-standard–based normalization, with multi-point calibration curves for key metabolites
LOD / LOQ	Typically in the low nanomolar (nM) to high micromolar (μM) range, depending on metabolite and sample type
Reproducibility	Intra- and inter-batch coefficient of variation (CV) typically <10–15% for most metabolites
QC strategy	Includes pooled QC samples, blanks, random injection order, technical replicates, and signal drift checks
Batch capacity	Flexible batch sizes, suitable for both pilot studies and large-cohort projects
Data output formats	Raw MS files (optional), integrated peak areas, normalized data, and concentration tables where applicable

Agilent 1260 Infinity II HPLC (Figure from Agilent)

Agilent 6495C Triple quadrupole (Figure from Agilent)

Orbitrap Exploris 240 (Figure from Thermo)

How Our Glycolysis Metabolomics Service Works

1. Consultation & Custom Panel Design

We clarify your research questions (e.g., glycolytic flux, PPP involvement, energy status) and recommend or customize a glycolysis-focused panel accordingly.

2. Sample Preparation

You collect tissues, cells, body fluids, or microbial samples following our SOPs; rapid quenching, snap-freezing, and storage at −80 °C are required to preserve labile glycolytic intermediates.

3. LC–MS/MS Analysis

Samples are analyzed by targeted LC–MS/MS with chromatographic separation of key isomers (e.g., G6P/F6P), internal standards, and MRM methods to ensure sensitive and reproducible quantification.

4. Data Processing & Statistical Analysis

We integrate peaks, normalize data, and perform statistical and pathway analysis (e.g., group comparisons, PCA/PLS-DA) to reveal glycolysis- and PPP-related metabolic shifts.

5. Results & Reporting

You receive processed data, key figures, and a concise methods and QC summary, with optional discussion to help link metabolic changes to your experimental model.

LC-MS/MS Workflow for Glycolysis Analysis

Typical Research Applications of Glycolysis Metabolism Analysis

Cancer Metabolism and Metabolic Reprogramming

Characterize glycolytic reprogramming (e.g., Warburg-like shifts) in tumor cell lines and animal models.
Compare glycolytic profiles across genotypes, environmental conditions (e.g., hypoxia), or metabolic gene perturbations.

Immunometabolism and Inflammation

Profile glycolysis during T cell activation, differentiation, or exhaustion.
Study how macrophage polarization (M1/M2) and other immune cell states depend on glycolytic flux and lactate production.

Metabolic Diseases, Obesity, and Diabetes Models

Assess glycolytic intermediates in diet-induced obesity, insulin resistance, and NAFLD models.
Evaluate how nutritional interventions, exercise protocols, or research compounds reshape glycolysis in liver, muscle, adipose tissue, and serum.

Microbial Metabolism and Fermentation Engineering

Quantify glycolytic intermediates and related pathways in bacterial and yeast strains under different culture or fermentation conditions.
Support strain engineering and process optimization by monitoring carbon flux and byproduct formation.

Cellular Bioenergetics, Stem Cells, Neurobiology, and Aging

Investigate how glycolysis and ATP/NAD(H) balance support stem cell maintenance, differentiation, and reprogramming.
Explore glycolytic contributions to neuronal and glial metabolism, muscle physiology, or age-associated metabolic remodeling.

Preclinical Compound Mechanism and Safety Assessment

Examine how small molecules, biologics, or genetic tools modulate glycolytic pathways in cell and animal models.
Use glycolysis-focused profiles to understand metabolic mechanisms, off-target effects, and systems-level responses in preclinical research.

Sample Submission Guidelines for Glycolysis Metabolomics Service

Sample Type	Recommended Amount	Container Requirements	Storage Conditions	Special Notes
Tissue	≥ 20 mg wet weight per sample	1.5 mL or 2.0 mL microcentrifuge tube (pre-chilled)	Immediately snap-freeze in liquid nitrogen; store at −80 °C	Avoid blood contamination; DO NOT use formalin-fixed or paraffin-embedded tissue
Adherent cells	≥ 1 million cells per sample (cell pellet)	1.5 mL microcentrifuge tube	Snap-freeze pellet after washing with cold PBS; store at −80 °C	Provide cell count and treatment info; avoid serum carryover
Suspension cells	≥ 1 million cells per sample (pelleted)	Same as above	Same as above	Pre-wash with cold PBS to remove media components
FACS-sorted cells	≥ 50,000–100,000 cells (depending on target)	Low-retention microtube	Freeze immediately in dry ice or liquid nitrogen	Consult us in advance; ultra-low volume protocol available
Serum / Plasma	≥ 100 μL per sample	0.5–1.5 mL cryovial (low-binding recommended)	Aliquot and freeze at −80 °C immediately after separation	Avoid repeated freeze–thaw cycles
Urine	≥ 500 μL	Same as above	Store at −80 °C	First-morning urine preferred; avoid preservatives
CSF	≥ 50 μL	Low-binding cryovial	Store at −80 °C	Handle with care; avoid hemolysis
Microbial pellets	From OD600 ~1.0 culture, ≥ 1 mL culture volume	1.5 mL tube	Pellet cells, wash once in cold PBS, freeze at −80 °C	Indicate strain, media, and growth phase
Fermentation broth / culture supernatant	≥ 1 mL (filtered or centrifuged)	Cryovial	Store at −80 °C	Clarify time point and medium used

Deliverables: What You Receive from GAG Analysis

Normalized metabolite data (peak areas, optional concentrations)
Group comparisons with fold change and p-values
PCA, PLS-DA, heatmaps, volcano plots
Pathway maps for glycolysis and related routes
Technical summary and QC metrics
Sample metadata (ID, type, treatment info)
Optional: raw MS files and custom visualizations

Chromatograms of G6P and F6P showing clear LC–MS/MS separation.

LC–MS/MS chromatograms showing baseline-separated G6P and F6P isomers with labeled MRM transitions.

Score plot showing metabolic separation between experimental groups.

PCA/PLS-DA score plot illustrating group separation based on glycolysis metabolite profiles with 95% confidence ellipses.

Pathway diagram of glycolysis and PPP with fold change overlay on metabolites.

Glycolysis and PPP pathway map with metabolite nodes colored by fold change (red up, blue down).

Plots showing group differences in glycolytic metabolites and energy ratios.

Bar/box plots of key glycolytic metabolites and ATP/ADP and NAD⁺/NADH ratios across experimental groups with significance marks.

How does your service distinguish between G6P and F6P isomers?

Glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) are isobaric, so mass spectrometry alone cannot separate them. We use optimized hydrophilic interaction liquid chromatography (HILIC) to achieve baseline chromatographic separation before MS detection. This ensures independent, accurate quantification of G6P and F6P, rather than a merged signal.

Why is LC–MS/MS superior to colorimetric or fluorometric assay kits for glycolysis analysis?

Assay kits are useful for single readouts (e.g., lactate) but are limited in specificity, multiplexing, and pathway coverage. Our LC–MS/MS platform simultaneously quantifies multiple glycolytic intermediates (e.g., G6P, F1,6BP, PEP, pyruvate, lactate) and energy cofactors (ATP/ADP), enabling pathway-level interpretation, flux ratios, and identification of enzymatic bottlenecks that single-analyte kits cannot provide.

Can this platform quantify energy charge (ATP/ADP/AMP) and redox state (NAD⁺/NADH)?

Yes. Our targeted panels can include ATP, ADP, AMP, NAD⁺, and NADH. We use dedicated extraction protocols to preserve these labile cofactors, allowing you to directly link glycolytic changes with cellular energy charge and redox status.

Why is "metabolic quenching" required for glycolysis samples?

Glycolytic intermediates and energy carriers turn over within seconds. Without rapid quenching, ongoing enzymatic activity distorts metabolite levels. We provide SOPs for immediate quenching (e.g., liquid nitrogen or cold organic solvents) to “freeze” the metabolic state at the time of sampling

What are the minimum sample requirements for rare cell populations (e.g., FACS-sorted cells)?

For standard workflows we recommend ~1×10⁶ cells per sample, but using our low-input protocols we can typically work with 50,000–100,000 cells (e.g., specific T-cell subsets, stem cells). Please discuss available cell numbers with us during the consultation so we can adjust extraction volumes accordingly.

Can I use formalin-fixed paraffin-embedded (FFPE) tissues for this service?

No. FFPE processing removes most polar metabolites and damages labile intermediates, making it unsuitable for glycolysis-focused metabolomics. We require fresh-frozen tissues, snap-frozen in liquid nitrogen and stored at −80 °C.

Is the data provided as absolute concentration or relative abundance?

By default, we provide normalized peak areas for robust group comparisons. Absolute quantification can be offered for selected metabolites using multi-point calibration and stable isotope–labeled internal standards.

How do you handle the high variability often seen in glucose and lactate levels?

We reduce variability using isotopically labeled internal standards, strict QC, and drift correction. In addition to glucose and lactate, we recommend focusing on intracellular intermediates (e.g., F1,6BP, PEP, 6-PG), which provide a more stable and informative readout of glycolytic and PPP flux.

Macrophage-associated lipin-1 promotes β-oxidation in response to proresolving stimuli

Schilke, R. M., Blackburn, C. M., Rao, S., Krzywanski, D. M., Finck, B. N., & Woolard, M. D.

Journal: Immunohorizons

Year: 2020

DOI: https://doi.org/10.4049/immunohorizons.2000047

Mechanisms underlying neonate-specific metabolic effects of volatile anesthetics

Stokes, J., Freed, A., et al.

Journal: eLife

Year: 2021

DOI: https://doi.org/10.7554/eLife.65400

Insulin resistance does not impair mechanical overload-stimulated glucose uptake, but does alter the metabolic fate of glucose in mouse muscle

Weyrauch, L. A., McMillin, S. L., & Witczak, C. A.

Journal: International Journal of Molecular Sciences

Year: 2020

DOI: https://doi.org/10.3390/ijms21134715

YAP mediates compensatory cardiac hypertrophy through aerobic glycolysis in response to pressure overload

Kashihara, T., Mukai, R., Oka, S. I., Zhai, P., Nakada, Y., Yang, Z., ... & Sadoshima, J.

Journal: The Journal of Clinical Investigation

Year: 2022

DOI: https://doi.org/10.1172/JCI150595

Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF‐1α

Pelletier, A., Nelius, E., Fan, Z., Khatchatourova, E., Alvarado‐Diaz, A., He, J., ... & Stockmann, C.

Journal: EMBO Reports

Year: 2023

DOI: https://doi.org/10.15252/embr.202256156