Kennedy Pathway Metabolites Quantification Service

Why Quantify the Kennedy Pathway

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are dominant membrane phospholipids, and the Kennedy pathway (CDP-choline / CDP-ethanolamine) is a primary synthesis route supplying them. In many mechanism studies, the critical question is not simply whether PC/PE changes, but what drives the change: precursor availability, a block at a specific step, altered DAG utilization, or increased remodeling/turnover.

By quantifying step-specific intermediates (e.g., phosphocholine, phosphoethanolamine, CDP-choline, CDP-ethanolamine) together with PC/PE outputs (and optional DAG and isotope tracing), this service helps you:

• Identify enzymatic rate-limiting steps and metabolic flux bottlenecks across the Kennedy architecture.
• Deconvolute the dynamics of steady-state shifts: distinguishing biosynthetic attenuation from accelerated phospholipolysis/remodeling.
• Explain membrane-driven phenotypes (ER/mitochondria/lipid droplets, stress responses) with quantitative evidence
• Validate gene/compound mechanisms of action using direct pathway readouts
• Track process/condition effects (media/nutrients/time-course) with reproducible metrics

Kennedy Pathway Analysis Service Modules & Quantification Options

Kennedy Pathway Polar Intermediates

Targeted quantification of water-soluble intermediates to pinpoint the rate-limiting step(s) in PC/PE synthesis, including phosphorylated and CDP-activated intermediates.

PC/PE Outputs (Targeted Lipidomics Panel)

Quantitative profiling of PC and PE to capture pathway outputs and membrane composition changes.

• Sum-composition profiling (e.g., PC 34:1, PE 36:2)
• MS/MS-confirmed species (fatty-acyl–resolved assignment where feasible)

Stable-Isotope Tracing

Isotopologue and label-incorporation analysis to distinguish reduced synthesis flux from increased turnover/remodeling, supporting time-course and perturbation studies.

Target List & Panel Coverage

Table 1. Polar Intermediates

Table 2. PC/PE Lipid Species Panel

We report PC/PE as sum compositions (total carbons : total double bonds), which is standard for targeted phospholipid profiling.

Optional lipid add-ons (common in ER/mitochondria projects):

• PE plasmalogens: PE(P-) / PE(O-) (panelized by sum composition)
• DAG species (sum composition set)
• LPC/LPE species panel

Advantages of Kennedy Pathway Metabolites Analysis Service

• Up to 10⁶ linear dynamic range for targeted quantification
  Modern tandem quadrupole systems commonly specify six orders of linear dynamic range for quantitative MRM/SRM workflows.
• High-sensitivity MRM suited for low-abundance intermediates
  Example capability (platform spec): QTRAP 6500+ reports 1 pg on-column reserpine at S/N > 1,500,000 in positive MRM.
• High-speed scheduled MRM ensuring >15 data points per peak even for dense panels
  Example capability: TSQ Altis class performance notes up to ~600 SRMs/sec, supporting robust scheduled MRM methods.
• Flux-ready option for mechanism clarity
  Stable-isotope tracing provides isotopologue distributions and label incorporation to interpret pathway activity rather than only steady-state abundance.
• Fit-for-purpose quantitation (absolute or relative)
  Choose absolute quantification (calibration curve-based) when cross-study comparability is needed, or relative quantification for screening/priority setting.

Workflow for Kennedy Pathway Metabolites Panel Analysis

1

Project scoping: research question → select modules → confirm target list and quant type (absolute/relative)

2

Study design support: replicates, randomization, QC placement; isotope tracer design (if Module C)

3

Sample receipt & batch plan: condition check, storage verification, run order & QC schedule

4

Extraction + internal standards: optimized workflows for polar intermediates and lipids

5

LC–MS/MS acquisition: scheduled MRM/SRM with appropriate polarity switching

6

Quantification & QC review: calibration, IS correction, carryover checks, QC drift evaluation

7

Reporting & delivery: final tables + QC package + method details + raw files

Analytical Platform (LC–MS/MS) & Key Parameter

Primary Platform

Triple Quadrupole LC–MS/MS for targeted quantification (MRM/SRM): SCIEX QTRAP 6500+ (Scheduled MRM / MRM)

• Mass range: m/z 5–2000
• Polarity switching: ~5 ms
• Minimum dwell time: 1 ms
• High-throughput targeted acquisition suitable for dense panels (scheduled MRM)

Chromatography Setups (Representative)

• Polar intermediates (Module A): HILIC LC method with positive/negative switching as needed
• PC/PE lipids (Module B): UPLC–C18 method optimized for phospholipids

Exact column chemistries, gradients, and run times are finalized after we confirm your sample matrix and throughput goals.

Optional (Upon Request)

If your study requires additional confirmation or expanded profiling, we can discuss alternative LC–MS configurations (e.g., high-resolution MS) based on project goals and sample matrix.

ExionLC AD System (Figure from Sciex)

Triple Quad™ 6500+ (Figure from Sciex)

TSQ Altis Triple Quadrupole MS (Figure from Thermo Scientific)

Sample Requirements for Kennedy Pathway Quantification

General recommendations

• Biological replicates: ≥ 3 per group (more recommended for subtle effects)
• Packaging: Seal tubes with parafilm; place in secondary sealed bag; include sample manifest (ID, matrix, group, volume/mass, storage history)
• Stability: Keep samples frozen at all times; avoid thaw/refreeze cycles

What You Receive (Deliverables)

Representative MRM chromatograms for Kennedy intermediates showing clean peaks and low background.

Calibration curves with wide linear range and LLOQ summary for absolute quantification.

Pooled QC stability across run order and CV distribution demonstrating batch-level reproducibility.

Kennedy pathway map overlaying log2 fold changes and q-values for mechanism-ready interpretation.

Research Applications: Kennedy Pathway PC/PE Synthesis Quantification

Membrane lipid homeostasis (PC/PE synthesis)

Targeted LC–MS/MS quantification of PC/PE balance and pathway intermediates.

ER & lipid droplet biology

Link phenotypes to Kennedy pathway capacity vs lipid remodeling/turnover.

Mitochondria-related membrane studies (PE-focused)

Quantify CDP-ethanolamine branch intermediates and PE output shifts.

Gene perturbation & pathway bottleneck mapping

Identify rate-limiting steps via phosphocholine/CDP-choline and related intermediates.

Compound MoA & pathway engagement

Confirm on-pathway effects; optional stable isotope tracing for flux vs turnover.

Nutrient/media & time-course studies

Track choline/ethanolamine utilization dynamics with quantitative readouts.

Phosphate Limitation Drives Phospholipid Remodeling in a Host–Symbiont System

Background

Reef-building cnidarians rely on intracellular photosynthetic symbionts, and maintaining a stable symbiosis requires tight coordination of host and algal cell division. While nitrogen control has been widely discussed, this study tested whether phosphate availability can act as a key regulator—linking nutrient status to membrane phospholipid pools and, ultimately, symbiont proliferation.

Samples:

• Symbionts grown in culture under phosphate-replete vs phosphate-depleted conditions
• Freshly isolated symbionts from the host for comparison to in vitro states

Technical Methods (Study Summary)

• Phosphate status characterization comparing in-host vs in vitro conditions
• Transcriptomics (RNA-seq) to evaluate phosphate-responsive programs
• Lipid profiling to assess membrane lipid changes under phosphate limitation

Results

• Freshly isolated symbionts exhibited a phosphate-limited state comparable to phosphate-starved cultures.
• Phosphate limitation was associated with broad lipid changes, including reduced phospholipid abundance, supporting a nutrient-to-membrane link in symbiont cell division control.

How Our Service Supports Similar Projects

Studies like this often identify "phospholipids change" as a key signature, but mechanism interpretation benefits from step-resolved quantification and species-level readouts. Our Kennedy Pathway Metabolites Quantification (Targeted LC–MS/MS) can be used to strengthen conclusions by addressing:

• Which synthesis branch is constrained?
  Polar Intermediates quantifies pathway intermediates such as phosphocholine / CDP-choline and phosphoethanolamine / CDP-ethanolamine, enabling bottleneck localization (precursor limitation vs CDP-activation step constraints).
• Which membrane outputs shift?
  PC/PE Outputs quantifies PC/PE species (sum composition; optional MS/MS-confirmed species) to reveal how membrane composition changes under phosphate limitation.
• Is the change reduced synthesis or increased turnover?
  Optional Stable-Isotope Tracing provides isotopologue/label incorporation outputs to separate flux reduction from remodeling/turnover-driven differences (fit for time-course or nutrient-shift experiments).

Reference

1. Faulstich, N. G., et al. "Evidence for phosphate-dependent control of symbiont cell division in the model anemone Exaiptasia diaphana." mBio (2024).