Purines Analysis Service

Overview of Purines

Purines are nitrogen-containing heterocyclic compounds that serve as fundamental building blocks in various biological systems. As core components of nucleotides—including adenosine monophosphate (AMP), guanosine monophosphate (GMP), and their corresponding deoxy forms—they are essential to DNA and RNA structure. Beyond their genetic role, purines also participate in critical cellular functions, including ATP-mediated energy transfer, GTP signaling, and cofactor synthesis (e.g., NAD⁺, FAD). Abnormal purine metabolism is closely linked to diseases such as gout, cancer, metabolic syndromes, and neurodegenerative disorders.

Why Analyze Purines?

Purine metabolism plays a foundational role in cellular homeostasis, linking genetic material, energy currency, and signaling molecules. Dysregulation in purine pathways is implicated in a wide range of biological processes, including oxidative stress, immune activation, mitochondrial dysfunction, and renal overload. In both basic research and applied settings, purine profiling offers unique value across multiple domains:

• Cellular energy balance: AMP/ATP ratios reflect metabolic shifts under hypoxia or drug treatment
• Stress and degradation pathways: Accumulation of hypoxanthine or uric acid reveals oxidative load and xanthine oxidase activity
• Biomarker discovery: Specific purine derivatives can signal disease progression or pharmacodynamic effects
• Pathway characterization: Insights into salvage and de novo biosynthesis routes support metabolic engineering and therapeutic design
• Bioprocess optimization: Fine-tuning purine availability in cell cultures or microbial systems improves yield and system stability

Analyzing purines is not just about quantifying molecules—it's about decoding the dynamics behind cell fate, metabolic flux, and disease adaptation.

Purines Analysis Service Offered by Creative Proteomics

• Quantification of canonical purine nucleotides: ATP, ADP, AMP, GTP, GDP, GMP across biological matrices (plasma, serum, CSF, tissue, cell lysate, microbial extracts)
• Profiling of purine bases and degradation products: adenine, guanine, hypoxanthine, xanthine, uric acid, inosine
• Measurement of purine nucleosides and nucleotides in dynamic time-course studies, including stress, hypoxia, and drug perturbation experiments
• Assessment of purine salvage and de novo synthesis pathway intermediates, enabling upstream/downstream pathway reconstruction
• Stable isotope-labeled tracing services (e.g., ¹³C-adenine, ¹⁵N-inosine) to monitor flux through purine biosynthetic pathways
• Comparative analysis of purine metabolism under different experimental conditions, such as disease models, engineered strains, or therapeutic interventions
• Custom method development for rare purine analogs, cofactor-associated species (e.g., NAD⁺, FAD), or pharmaceutical derivatives

List of Detected Purines and Related Metabolites

Advantages of Purines Assay

• High Sensitivity and Low Detection Limits: Achieve detection down to 0.1–0.5 ng/mL for key purine metabolites such as ATP, hypoxanthine, and uric acid, suitable for low-abundance biological samples (e.g., CSF, intracellular extracts).
• Excellent Quantitative Precision: Intra- and inter-assay CV ≤ 5%, ensuring consistent results across replicates and batches.
• Wide Dynamic Range: Linear quantification across four to five orders of magnitude (e.g., 0.1 ng/mL to 100 µg/mL), enabling simultaneous detection of trace and abundant analytes.
• Pathway Coverage: Simultaneous analysis of 20+ purine-related compounds across de novo synthesis, salvage, and degradation pathways in a single run.
• Multiplexed Sample Processing: Process up to 96 samples per batch with automated extraction and LC-MS/MS quantification, optimizing throughput for cohort studies or time-course designs.
• Custom Ratio Calculations & Pathway Indices: Support for metabolic indices such as ATP/AMP, IMP/XMP, hypoxanthine/uric acid, tailored to disease modeling or stress profiling.
• Stable Isotope Tracing Support: Integrated workflows using ¹³C- or ¹⁵N-labeled compounds enable pathway-specific flux quantification and kinetic studies.

Workflow for Purines Analysis Service

Technology Platform for Purines Analysis Service

LC-MS/MS: Thermo Scientific™ TSQ Quantis™ Triple Quadrupole for targeted quantification

UHPLC: Agilent 1290 Infinity II for high-resolution chromatographic separation

Data Handling: Thermo Xcalibur™ and Compound Discoverer™ for signal processing and identification

Optional: Stable isotope tracing workflows available using ¹³C or ¹⁵N-labeled purines

TSQ Quantis™ Triple Quadrupole (Figure from Thermo Scientific)

Agilent 1260 Infinity II HPLC (Fig from Agilent)

Sample Requirements for Purines Analysis Service

Demo Results

Concentrations of the detected compounds were calculated with internal-standard calibration by interpolating the constructed linear calibration curves of individual compounds within an appropriate concentration range for each compound.

Table 1 . Quantitative Results of Purine-Related Compounds in Test and QC Samples

Purines Analysis Service Case Study

Title: Comprehensive quantification of purine and pyrimidine metabolism in Alzheimer's disease postmortem cerebrospinal fluid by LC–MS/MS with metal-free column
Journal: Biomedical Chromatography
Published: 2018

• Background
• Methods
• Results
• Reference

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and behavioral disturbances in the elderly. Pathologically, it involves neurofibrillary tangles of tau protein and amyloid β plaques. Beyond these, mitochondrial dysfunction, neuroinflammation, and neurotransmitter disruptions are implicated in its progression. Purine and pyrimidine metabolism, critical for energy transfer and cellular regulation, have been linked to AD pathology.

The study employed liquid chromatography with tandem mass spectrometry (LC–MS/MS) for comprehensive analysis of 40 nucleobases, nucleosides, nucleotides, and phosphorylated metabolites involved in purine and pyrimidine metabolic pathways, typical of metabolomics studies. A metal-free PEEK column was utilized to overcome chromatographic challenges associated with interactions between phosphorylated residues and metal surfaces, which typically lead to peak tailing and reduced sensitivity.

The optimized LC–MS/MS method utilized a mobile phase of pH 7 with 10 mM ammonium formate to enhance stability and sensitivity, essential for metabolite detection in complex biological samples. Injection volume was set at 2 μl to ensure optimal peak shape and avoid double-peaking issues observed at higher volumes. Gradient elution conditions were tailored to achieve efficient separation and cleaning of the column, crucial for maintaining consistent performance and minimizing carryover in metabolomics analyses.

Analytical performance validation demonstrated the method's robustness: calibration curves exhibited excellent linearity (r² > 0.9959) across a concentration range of 5 nM to 0.1 mM for all compounds. The method achieved low limits of detection (LOD) ranging from 0.5 to 250 nM and limits of quantification (LOQ) ranging from 1 to 500 nM, surpassing those reported for ion-pairing LC–MS/MS methods (Laourdakis, 2014). Recovery rates for most compounds ranged from 70.5% to 144% in 10-fold diluted postmortem cerebrospinal fluid (pCSF) samples, indicating satisfactory accuracy for quantification despite matrix effects.

Application of the method to pCSF samples from control subjects and AD patients revealed detectable levels of 15 targeted compounds above LOQ values, predominantly downstream metabolites of purine and pyrimidine pathways. Statistical analysis, while showing variability among individual samples, indicated potential differences in nucleotide concentrations between control and AD groups, highlighting the method's utility in biomarker discovery and disease mechanism elucidation.

Reference

1. Muguruma, Yoshio, et al. "Comprehensive quantification of purine and pyrimidine metabolism in Alzheimer's disease postmortem cerebrospinal fluid by LC–MS/MS with metal‐free column." Biomedical Chromatography 34.2 (2020): e4722. https://doi.org/10.1002/bmc.4722