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Measuring protein synthesis by mass isotopomer distribution analysis (MIDA)
C Papageorgopoulos1, K Caldwell, C Shackleton
1Department of Nutritional Sciences, University of California at Berkeley, Berkeley, California, 94720, USA.
Analytical Biochemistry
|January 27, 1999
Summary
Mass isotopomer distribution analysis (MIDA) now accurately measures protein synthesis and precursor pools using peptides and mass spectrometry. This stable isotope technique overcomes previous limitations for in vivo studies.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Molecular Biology
Background:
- Measuring protein kinetics using isotopic techniques is challenging due to difficulties in quantifying the aminoacyl-tRNA precursor pool.
- Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometry (MS) method for determining precursor enrichments from polymeric products.
- Analyzing complex protein isotopomer patterns by MS is analytically demanding.
Purpose of the Study:
- To describe and validate a novel approach for measuring protein synthesis using MIDA.
- To assess the feasibility of MIDA for determining precursor pool enrichments in vitro and in vivo.
- To evaluate the accuracy and precision of MIDA with different mass spectrometry instruments.
Main Methods:
- Synthesized peptides using known mixtures of natural abundance and labeled leucine for in vitro studies.
- Simulated in vivo biosynthesis by mixing enriched and natural abundance peptides.
- Analyzed synthetic and biological peptide mass isotopomer patterns using electrospray ionization mass spectrometry (ESI-MS) with magnetic sector and quadrupole analyzers.
- Infused rats with labeled leucine and analyzed a leucine-rich peptide from serum albumin via ESI-MS.
Main Results:
- MIDA successfully resolved mass isotopomer patterns for synthetic peptides with comparable accuracy and precision between magnetic sector and quadrupole instruments.
- In vivo analysis of rat serum albumin yielded fractional synthetic rates (0.45 ± 0.03 day⁻¹) consistent with published data.
- The study demonstrated the technical feasibility and practicality of MIDA for in vivo protein synthesis measurement.
Conclusions:
- Measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practical in vivo.
- Proteolytically derived peptides coupled with ESI-MS analysis are effective for MIDA applications.
- This method overcomes previous limitations in measuring isotope content of the biosynthetic precursor pool.