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Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer
Published on: August 2, 2018
Motif-specific sampling of phosphoproteomes
Cristian I Ruse1, Daniel B McClatchy, Bingwen Lu
1Department of Chemical Physiology/Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Journal of Proteome Research
|May 3, 2008
Summary
This study introduces a novel phosphoproteomics method using Barium ion (Ba2+) binding to identify motif-specific phosphopeptides. This technique enhances the coverage of the phosphoproteome, aiding cell signaling research.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Phosphoproteomics is crucial for understanding cell signaling dynamics.
- Current methods may have limitations in comprehensive phosphopeptide identification.
Purpose of the Study:
- To develop and validate a novel methodology for phosphoproteome and proteome analysis.
- To enhance the identification and coverage of motif-specific phosphopeptides.
- To investigate cell signaling pathways through quantitative phosphoproteomics.
Main Methods:
- A Ba2+ binding-based methodology was developed to link phosphoproteome and proteome analysis.
- Multidimensional Identification Technology (MudPIT) was employed for phosphopeptide identification.
- Nuclear extracts from HeLa cells and stimulated HEK cells were analyzed.
Main Results:
- The method identified 1037 phosphopeptides from 250 μg of protein.
- Over 70% of identified phosphoproteins were confirmed by their nonmodified peptides.
- Quantitative analysis revealed changes in protein and phosphorylation levels for 197 phosphoproteins, including MAPK1, upon stimulation.
Conclusions:
- The Ba2+ binding-based approach offers a robust method for motif-specific phosphopeptide selection.
- This technique significantly extends phosphoproteome coverage and aids in dissecting signaling pathway dynamics.
- Integration of quantitative phosphoproteomics data with pathway knowledge enhances understanding of kinase roles in cellular signaling.
