Quantitative Glycan Metabolism & Pathway Flux Analysis

Why Measure Glycan Metabolism?

Glycosylation is tightly regulated by metabolic input—not just enzyme activity. Key pathways like the Hexosamine Biosynthesis Pathway (HBP) integrate glucose, glutamine, acetyl-CoA, and UTP to produce UDP-GlcNAc, the essential donor for O-GlcNAcylation and N-glycan branching. Elevated HBP activity is a hallmark of metabolic reprogramming in cancer and immune activation.

Likewise, levels of CMP-Sialic Acid directly control the cell's capacity for sialylation, a key modification in immune evasion and metastasis. These and other nucleotide sugars function as metabolic checkpoints that link nutrient availability to glycan output.

Quantifying these intermediates provides a sensitive, upstream, and mechanistic view of glycosylation changes, often revealing biological shifts earlier than structural glycan profiling.

What We Quantify: High-Coverage, Targeted Panels for Glycan Metabolism

Creative Proteomics offers curated, LC-MS/MS–based panels targeting key metabolites across glycan biosynthesis and nucleotide sugar metabolism. These panels are designed to quantify metabolic inputs relevant to N- and O-glycosylation, sialylation, fucosylation, and glycosaminoglycan (GAG) biosynthesis.

Our platform targets nucleotide sugars, pathway intermediates, and metabolic cofactors—ensuring decision-ready data for mechanistic studies, disease models, and drug development.

Representative Metabolites in Glycan Biosynthesis Pathways

Custom panels are available. Please inquire if your project requires specific pathway branches or rare intermediates.

Pathway Flux Analysis: Measuring Metabolic Activity Through Isotope Tracing

To investigate not just metabolite abundance but how actively pathways are functioning, we support Stable Isotope Tracing (SIT) using ¹³C- or ¹⁵N-labeled substrates. This approach enables quantitative analysis of flux through glycan-related pathways by tracking label incorporation into downstream metabolites.

By analyzing the isotopic distribution of compounds such as UDP-GlcNAc, CMP-Neu5Ac, or other nucleotide sugars, researchers can assess de novo synthesis rates, substrate utilization, and pathway dynamics under different biological or experimental conditions.

What the Service Includes

• Experimental Setup Support
  We assist in tracer selection, labeling duration, and sampling timing based on the turnover characteristics of your target pathway.
• Isotopologue Analysis via High-Resolution LC-MS/MS
  Orbitrap instrumentation allows precise quantification of labeled isotopologues across multiple metabolic intermediates.
• Data Interpretation and Reporting
  Results include isotopologue distributions, labeling patterns, and fractional enrichment, which can be used to infer relative pathway activity or response to perturbation.

Why Choose Our Glycan Metabolism & Pathway Flux Analysis?

• Ultra-sensitive quantification — Detect nucleotide sugars and intermediates down to 0.5–10 nM.
• High linearity and recovery — Calibration R² ≥ 0.995, recovery 85–115%, ensuring true absolute values.
• Dual-instrument precision — Agilent 6495C QQQ for quantitation + Orbitrap HF-X for isotopologue flux analysis.
• Reproducible performance — Inter- and intra-batch CV ≤ 15% for confident multi-sample comparison.
• Flux-ready analytics — Quantify ¹³C/¹⁵N label incorporation to reveal real-time pathway activity.
• Matrix-validated methods — Optimized extraction and calibration for cells, tissues, and biofluids.
• Integrated interpretation — Deliverables include isotopologue distribution + KEGG-mapped pathway insights.
• Actionable biological insight— Translate metabolic data into clear evidence of glycosylation regulation.

Technology & Validation for Glycan Metabolite Quantification

Our glycan metabolism and flux analysis services are built on validated LC-MS/MS platforms with stringent quality control procedures. We apply a dual-instrument strategy optimized for both absolute quantification and isotopologue resolution:

• Agilent 6495C Triple Quadrupole (QQQ) in dynamic MRM mode is used for targeted quantification, offering excellent sensitivity and linearity across wide dynamic ranges.
• Thermo Q Exactive™ HF-X Hybrid Orbitrap is employed for high-resolution isotopologue detection and discovery-based analysis in flux studies.
• Specialized Chromatography: HILIC (amide-based) and Porous Graphitic Carbon (PGC) methods are selected based on analyte polarity and structural similarity, particularly for isomeric nucleotide sugars.

Method Performance and Quality Specifications

Agilent 6495C Triple Quadrupole (Figure from Agilent)

Agilent 1260 Infinity II HPLC (Fig from Agilent)

Workflow for Quantitative Glycan Metabolism Analysis

1

Project Scoping — Define objectives, target pathways/analytes, matrices, and (if applicable) isotope tracing strategy.

2

Method Fit & Panel Finalization — Confirm analyte list, internal standards, calibration range, and acceptance criteria.

3

Sample Preparation & Shipment — Follow metabolomics SOPs; freeze promptly and ship on dry ice to maintain integrity.

4

Extraction & Batch QC — Cold-chain extraction with process blanks, matrix QCs, and calibration standards in each batch.

5

Targeted LC-MS/MS Acquisition — dMRM/PRM acquisition with isotope-labeled internal standards; routine system suitability checks.

6

Reporting & Review — Deliver quantitative tables, QC metrics, and (if applicable) isotopologue data for flux interpretation; optional consult for data review.

Sample Requirements for Glycan Metabolism LC-MS/MS Analysis

Notes: Use low-bind tubes; keep materials pre-chilled; record time-to-quench; label with Project/Sample ID & date; provide normalization data (preferred: protein by BCA; alternatives: cell count or tissue weight). For SIT, include tracer type, enrichment, and labeling duration.

What You Receive: Deliverables from Our Glycan Metabolism Analysis

• Quantitative concentration table: All analytes, sample IDs, final units (μM or nmol/mg protein).
• Full QC report: L/M/H QCs, calibration (R²), %RSD, accuracy, retention time windows, and acceptance criteria.
• Flux analysis report (if applicable): Isotopologue distributions, fractional enrichment, and pathway-specific flux visuals.
• Methodology summary: A "Materials & Methods" section ready for manuscripts and regulatory documentation.
• Raw data on request: .raw or .mzML files for archival and independent review.

Co-elution & Selectivity (Single EIC Overlay)

High-Resolution Product-Ion Spectrum (Single MS/MS Spectrum)

Research Applications of Glycan Metabolism Quantification

Cancer Cell Biology

Quantify nucleotide-sugar pools to map glycosylation-relevant metabolic rewiring.

Immunometabolism

Track precursor availability during immune cell activation and phenotype transitions.

Neuroscience Research

Assess UDP-GlcNAc dynamics to study O-GlcNAc–linked neuronal regulation.

Stem Cell & Differentiation

Monitor pathway precursors across lineage commitment and maturation.

Bioprocess & Cell Culture Optimization

Profile media-dependent impacts on glycan-related metabolism in vitro.

Enzymology & Chemical Biology 

Evaluate pathway perturbations to probe glycosylation enzyme mechanisms.