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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Quantitative proteomic analysis to profile dynamic changes in the spatial distribution of cellular proteins
Wei Yan1, Daehee Hwang, Ruedi Aebersold
1Institute for Systems Biology, Seattle, WA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 29, 2008
Summary
This study introduces a new method for simultaneously profiling multiple organelles within a single cell lysate using quantitative proteomics. This approach overcomes limitations of traditional methods, enabling better understanding of dynamic protein localization and organelle function.
Area of Science:
- Cell Biology
- Proteomics
- Biochemistry
Background:
- Traditional organelle protein profiling relies on subcellular fractionation and mass spectrometry (MS), often yielding impure organelle isolates.
- Difficulty in isolating pure organelles and analyzing proteins that shuttle between them limits understanding of cellular localization.
- Dynamic protein movement between organelles necessitates concurrent analysis of multiple organelles from a single cell lysate.
Purpose of the Study:
- To develop an integrated experimental approach for simultaneous profiling of multiple organelles.
- To overcome the limitations of traditional subcellular fractionation methods.
- To enable the investigation of dynamic changes in organelle protein composition under various physiological conditions.
Main Methods:
- Subcellular fractionation of cell lysates via density gradient centrifugation.
- Isobaric tag for relative and absolute quantitation (iTRAQ) labeling for quantitative proteomic analysis.
- Mass spectrometry (MS) analysis of fractionated proteins and principal component analysis (PCA) for data interpretation.
Main Results:
- Simultaneous quantitative proteomic profiling of multiple organelles (ribosome, mitochondria, proteasome, lysosome, ER, Golgi) from a single assay.
- Acquisition of quantitative signature patterns for various organelles.
- Application of comparative PCA to investigate organelle profile changes between control and perturbed cellular conditions.
Conclusions:
- The developed integrated approach enables simultaneous profiling of multiple organelles, enhancing the accuracy of cellular localization studies.
- This quantitative proteomics-based method provides a powerful tool for dissecting cellular protein organization and detecting dynamic changes in organelle composition.
- The method facilitates the investigation of organelle dynamics and protein functional units in response to different physiological states.
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