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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
An automated multidimensional protein identification technology for shotgun proteomics.
D A Wolters1, M P Washburn, J R Yates
1Torrey Mesa Research Institute, San Diego, California 92121, USA.
Analytical Chemistry
|January 5, 2002
Summary
We developed an automated shotgun proteomics method called multidimensional protein identification technology (MudPIT). This technique enhances proteome analysis by improving sample preparation and separations for identifying diverse proteins.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Shotgun proteomics enables large-scale protein identification.
- Previous methods faced limitations in sensitivity and throughput.
- Automated, high-throughput proteomic analysis is crucial for biological research.
Purpose of the Study:
- To introduce an automated method for shotgun proteomics.
- To improve upon existing multidimensional liquid chromatography techniques.
- To enhance the identification of proteins within complex mixtures.
Main Methods:
- Developed multidimensional protein identification technology (MudPIT).
- Integrated strong cation-exchange (SCX) and reversed-phase resins in a biphasic column for liquid chromatography.
- Utilized electrospray ionization tandem mass spectrometry for peptide analysis.
Main Results:
- Achieved high reproducibility (within 0.5%) in MudPIT analyses.
- Demonstrated a dynamic range of 10,000:1 for complex peptide mixtures.
- Improved the identification of proteins across all functional and physical classes.
Conclusions:
- MudPIT offers a robust and reproducible automated approach for shotgun proteomics.
- The method significantly enhances the depth and breadth of proteome analysis.
- This advancement facilitates comprehensive identification of proteins in complex biological samples.
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