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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
Published on: August 17, 2015
Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures
Y Shinohara1, Y Suzuki, H Hasegawa
1Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan. sinohara@toyaku.ac.jp
Nucleosides, Nucleotides & Nucleic Acids
|December 3, 2011
Summary
This study presents a novel gas chromatography-mass spectrometry method to measure 5-Phosphoribosyl-1-pyrophosphate (PRPP) in red blood cells. The method accurately quantifies PRPP by measuring labeled glutamate formation, crucial for purine synthesis regulation.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Metabolic Regulation
Background:
- 5-Phosphoribosyl-1-pyrophosphate (PRPP) is a key regulator of de novo purine synthesis.
- Accurate measurement of PRPP is essential for understanding purine metabolism and related disorders.
- Existing methods for PRPP quantification may have limitations in sensitivity or specificity.
Purpose of the Study:
- To develop and validate a sensitive gas chromatographic-mass spectrometric (GC-MS) method for measuring PRPP in human erythrocytes.
- To establish a reliable assay for PRPP utilizing stable isotope labeling and enzymatic conversion.
- To provide a tool for clinical and research applications involving purine metabolism.
Main Methods:
- Erythrocytes were incubated with [(13)C(5)]glutamine and varying amounts of PRPP.
- PRPP utilization was measured by quantifying [(13)C(5)]glutamate formed via amidophosphoribosyltransferase.
- [(13)C(5)]glutamate was purified using anion-exchange chromatography and derivatized.
- Quantification was performed using GC-MS with [(13)C(2)]glutamate as an internal standard.
Main Results:
- The GC-MS method demonstrated good intra-day reproducibility for [(13)C(5)]glutamate determination.
- A linear correlation was observed between the amount of PRPP added and the amount of [(13)C(5)]glutamate produced.
- The method successfully quantified PRPP in erythrocyte samples.
Conclusions:
- The developed GC-MS method is a reliable and sensitive assay for measuring PRPP in erythrocytes.
- This assay facilitates the study of purine synthesis regulation and potential metabolic abnormalities.
- The method provides a valuable tool for biochemical research and clinical diagnostics.
