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


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.

  • Chrisler WB
  • Chu RK
  • Hsu CC
  • Jorgensen ML
  • Kitata RB
  • Liu T
  • Moore RJ
  • Rodland KD
  • Shi T
  • Smith RD
  • Tsai CF
  • Velickovic M
  • Wang YT
  • Wasserfall CH
  • Williams SM
  • Zhao R
  • Zhu Y
PubMed ID
Appears In
Commun Biol, 2023, 6 (1)