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A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
Published on: March 13, 2014
Optimizing identification and quantitation of 15N-labeled proteins in comparative proteomics
Joost W Gouw1, Bastiaan B J Tops, Peter Mortensen
1Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Analytical Chemistry
|September 24, 2008
Summary
This study addresses suboptimal heavy nitrogen ((15)N) enrichment in quantitative proteomics. We developed correction methods to improve protein identification and quantitation accuracy, even with enrichment below 100%.
Area of Science:
- Proteomics
- Biochemistry
- Analytical Chemistry
Background:
- Comparative proteomics utilizes stable isotopes for accurate protein quantification.
- Metabolic labeling with heavy nitrogen ((15)N) is widely used in organisms like Drosophila, C. elegans, and rats.
- Suboptimal (15)N enrichment (<98%) can negatively impact protein identification and quantification accuracy.
Purpose of the Study:
- To systematically investigate the impact of varying (15)N enrichment levels on protein identification and quantitation.
- To develop and validate correction methods for improving data quality in (15)N-based quantitative proteomics.
- To enhance the reliability of proteomic analyses using (15)N metabolic labeling with incomplete enrichment.
Main Methods:
- Analysis of two independent (15)N-labeled proteomic datasets.
- Investigation of peptide representation based on size and nitrogen isotope labeling.
- Development of computational methods to correct for under-representation and inaccurate peptide ratios.
- Validation of correction methods for improved protein quantitation.
Main Results:
- Larger (15)N-labeled peptides were found to be under-represented compared to their (14)N counterparts.
- A novel correction method significantly increased the number of protein identifications.
- A second method was developed to correct inaccurate peptide ratios caused by incomplete (15)N enrichment.
- The developed methods resulted in improved accuracy and precision in protein quantitation.
Conclusions:
- Incomplete (15)N enrichment poses challenges for quantitative proteomics.
- Correction methods can significantly enhance protein identification and quantitation accuracy.
- This study provides valuable tools for optimizing proteomic data quality in (15)N-labeled experiments with enrichment below 100%.
