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bioRxiv · 10.1101/2022.04.12.488038

A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Tsai, C.-F.·Wang, Y.-T.·Hsu, C.-C.·Kitata, R. B.·Chu, R. K.·Velickovic, M.·Zhao, R.·Williams, S. M.·Chrisler, W. B.·Jorgensen, M. L.·Moore, R. J.·Zhu, Y.·Rodland, K. D.·Smith, R. D.·Wasserfall, C. H.·Shi, T.·Liu, T.
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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 developed a novel 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 >3,000 (>9,500) phosphopeptides from 1 (10) {micro}g of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of [~]600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and [~]700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 {micro}m (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.

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Tsai, C.-F., Wang, Y.-T., Hsu, C.-C., Kitata, R. B., Chu, R. K., Velickovic, M., Zhao, R., Williams, S. M., Chrisler, W. B., Jorgensen, M. L., Moore, R. J., Zhu, Y., Rodland, K. D., Smith, R. D., Wasserfall, C. H., Shi, T., Liu, T.. 2022-04-12. A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics. https://doi.org/10.1101/2022.04.12.488038

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