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Home > Services > Metabolomics Service > Targeted Metabolomics > Nucleoside/Nucleotide Analysis Service

Nucleoside/Nucleotide Analysis Service

Nucleoside/Nucleotide Analysis Service

Nucleic acids are organic polymeric macromolecules composed of nucleotide monomers building blocks. A nucleotide is made of nucleobase, a five-carbon sugar and 1-3 phosphate groups. Though the nucleotide normally refers nucleoside monophosphate, now nucleoside diphosphate or nucleoside triphosphate are also belongs to nucleotides. The base in nucleotides can be either purine or pyrimidine. When the sugars are riboses, the nucleotides are ribonucleotides. While the sugars are deoxyriboses, the nucleotides are deoxyribonucleotides.

Nucleotides are the building blocks that are linked to form the nucleic acids ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), which act as the cell's storehouse of genetic information. Nucleotides play a central role in metabolism, serving as the convenient and universal carriers of metabolic energy and high-energy electrons in the form of the nucleoside triphosphates (ATP, GTP, CTP and UTP). ATP is the main source of the energy required by biosynthetic reactions. When hydrolyzed to ADP (adenosine diphosphate) or AMP (adenosine monophosphate), energy from ATP is released. When a reaction requiring energy is coupled to this energy release process, this reaction can occur. Because of this, ATP is commonly referred as the "energy currency of the cell." In addition, nucleotides are also important coenzymes, serving to carry chemical functional groups needed for enzyme activity such as coenzyme A (CoA), FAD, NAD and NADP+. Free nucleotides are also important in cellular signaling. The combination of hormone binds with the cell-surface receptor promotes the production of cyclic AMP (cAMP) inside the cell, which in turn elicit a wide variety of physiological and pathophysiological responses.

The release mechanisms of extracellular nucleotides and nucleosides involve in response to mechanical and chemical stimuli or pathophysiological states like inflammation and hypoxia. The extracellular nucleotides and nucleosides act at plasma membrane-bound purinoceptors (P1R and P2R) to elicit a series of downstream processes within the cell after release. The P1R family is divided into four receptor subtypes (A1, 2A, 2B, 3), whereas the P2R family is composed of seven P2X subunits (P2X1-7) that form homo- or heteromeric ion channels and eight P2Y subtypes (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11-14). Both P1R and P2R receptors are G protein-coupled receptors and involved in a wide variety of physiological and pathophysiological processes ranging from digestion to neurotransmission, immune response and pain sensation. What’s more, extracellular nucleotide and nucleoside signaling plays an important regulatory role in the kidney medullary microcirculation and renal collecting duct tubular transportation.

Historically, ATP concentration is measured with the established highly sensitive luciferin-luciferase luminescence assay technique appropriate for measuring localized ATP. Alternative analytical methods include HPLC-UV, LC-MS. While ATP is a useful biomarker for certain diseases, the significance of other nucleotides and nucleosides remains largely unexplored. Simultaneous quantification of ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP can’t be achieved with the luciferin-luciferase assay. To quantify up to 12 different extracellular nucleotides and nucleosides in samples of varying complexity, creative proteomics develop a simple, fast and reliable LC-MS method.

Platform

  • HPLC-UV

Summary

  • Identification and quantification of nucleotides by HPLC-UV

Sample Requirement

  • Normal Volume: 200ul serum/plasma; 200 mg tissue, 2e7 cells
  • Minimal Volume: 50ul serum/plasma; 50 mg tissue, 5e6 cells

Report

  • A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters
  • Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%
  • The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

Nucleotides Quantified in This Service

Nucleotides Quantified in This Service
1 Nicotinamide adenine dinucleotide (NAD)
2 Nicotinamide adenine dinucleotide,reduced (NADH)
3 Nicotinamide adenine dinucleotide phosphate (NADP)
4 Nicotinamide adenine dinucleotide phosphate, reduced (NADPH)
5 ADP
6 AMP
7 ATP
8 GDP
9 GTP

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective nucleotides targeted metabolomics services.


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