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Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics
Julian P Whitelegge1, Jonathan E Katz, Katianna A Pihakari
1The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry, University of California, Los Angeles, CA 90095, USA. jpw@chem.ucla.edu
Phytochemistry
|July 28, 2004
Summary
Subtle modification of isotope ratio proteomics (SMIRP) enables in vivo isotope coding for plants. Minor (13)C alterations do not impede proteomics software, but severe changes do, necessitating new deconvolution software.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Isotope ratio analysis is crucial for quantitative proteomics.
- Current methods face challenges in accurate sample coding and data interpretation.
- Understanding measurement variability is key to improving relative expression proteomics.
Purpose of the Study:
- To investigate the feasibility of using subtle modifications in isotope ratios for in vivo sample coding in proteomics.
- To assess the impact of altered (13)C abundance on peptide isotopic distribution and measurement variability.
- To evaluate the performance of proteomics software with modified isotope ratios and develop deconvolution software.
Main Methods:
- Minor alterations in (13)C abundance (approx. 1-2%) were applied to samples.
- Peptide isotopic distributions and inferred isotope ratios were analyzed.
- Performance of data-dependent acquisition and database searching software was tested.
- Development of Isosolv software for isotope ratio deconvolution alongside protein identification.
Main Results:
- A 2% increase in (13)C abundance caused measurable effects on peptide isotopic distribution and inferred isotope ratio.
- A 4% increase in (13)C abundance extended isotopic distribution and introduced background peaks.
- Subtle (1-2%) (13)C modifications did not affect proteomics software performance.
- More severe modifications significantly degraded software performance.
Conclusions:
- Subtle modification of isotope ratio proteomics (SMIRP) is a viable approach for in vivo isotope coding, particularly in plants.
- Understanding and controlling measurement variability is essential for accurate relative expression proteomics.
- The developed Isosolv software aims to improve isotope ratio decoding and protein identification in LC-MSMS.
- SMIRP may be extendable to mammals, offering a convenient in vivo coding strategy.