A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics.

Autor: Tsai CF; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA. chia-feng.tsai@pnnl.gov., Wang YT; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Hsu CC; Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan., Kitata RB; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Chu RK; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Velickovic M; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Zhao R; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Williams SM; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Chrisler WB; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Jorgensen ML; Department of Pathology, Immunology, and Laboratory Medicine, Diabetes Institute, College of Medicine, University of Florida, Gainesville, FL, 32611, USA., Moore RJ; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Zhu Y; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Rodland KD; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Smith RD; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA., Wasserfall CH; Department of Pathology, Immunology, and Laboratory Medicine, Diabetes Institute, College of Medicine, University of Florida, Gainesville, FL, 32611, USA., Shi T; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA. tujin.shi@pnnl.gov., Liu T; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA. tao.liu@pnnl.gov.
Jazyk: angličtina
Zdroj: Communications biology [Commun Biol] 2023 Jan 18; Vol. 6 (1), pp. 70. Date of Electronic Publication: 2023 Jan 18.
DOI: 10.1038/s42003-022-04400-x
Abstrakt: 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.
(© 2023. Battelle Memorial Institute, Hsu, Jorgenson, and Wasserfall.)
Databáze: MEDLINE
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