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Depth of proteome issues: a yeast isotope-coded affinity tag reagent study.
Kenneth C Parker1, Dale Patterson, Brian Williamson
1Discovery Proteomics and Small Molecule Research Center, Applied Biosystems, Framingham, MA 01701, USA. parkerkc@apliedbiosystems.com
Molecular & Cellular Proteomics : MCP
|March 30, 2004
Summary
This study optimized proteomics experiments using yeast, identifying ~700 proteins and improving confidence in peptide identification. Researchers developed informatics structures to characterize protein expression changes, especially for modified peptides.
Area of Science:
- Proteomics
- Yeast Molecular Biology
- Biochemistry
Background:
- Nonsense-mediated mRNA decay (NMD) pathway regulation is crucial in cellular processes.
- Optimizing proteomics experimental workflows is essential for accurate protein expression analysis.
Purpose of the Study:
- To test and optimize proteomics experimental procedures using a yeast model.
- To develop an informatics structure for characterizing protein identification and quantification.
- To assess the reproducibility of peptide identification in complex proteomic datasets.
Main Methods:
- Isotope-Coded Affinity Tag (ICAT) labeling for relative protein quantification.
- Matrix-Assisted Laser Desorption/Ionization (MALDI) and Electrospray (ESI) tandem mass spectrometry (MS/MS).
- Homologous recombination for UPF1 gene knockout in yeast.
Main Results:
- Approximately 700 proteins were identified with high confidence and quantified.
- Most proteins showed no significant expression change after UPF1 knockout.
- Reproducible changes observed in arginine biosynthesis proteins; UPF1 protein itself was downregulated.
- Artifacts in quantification due to overlapping heavy/light pairs were identified and addressed.
- Numerous chemically modified and non-tryptic peptides were identified, often corresponding to abundant proteins.
Conclusions:
- The study successfully optimized proteomics workflows, enhancing confidence in peptide identification.
- Developed informatics strategies improved the characterization of protein expression changes and identification confidence.
- The findings highlight the importance of addressing quantification artifacts and identifying modified peptides for accurate proteomic analysis.