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A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics.
- Source :
- Communications Biology; Dec2023, Vol. 6 Issue 1, p1-12, 12p
- Publication Year :
- 2023
-
Abstract
- Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level. A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics is developed, reducing sample loss and processing time, allowing the phosphoproteome profiling of mass-limited samples at the low nanogram level. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 23993642
- Volume :
- 6
- Issue :
- 1
- Database :
- Complementary Index
- Journal :
- Communications Biology
- Publication Type :
- Academic Journal
- Accession number :
- 161359884
- Full Text :
- https://doi.org/10.1038/s42003-022-04400-x