PNNL

Abstract

Effective nanoscale phosphoproteome analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during low stoichiometric phosphopeptide enrichment. The integration of a tandem tip sample manipulation method 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 identification of >3,000 (>9,500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. When integrated with the iBASIL method, it 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 at 200 µm × 200 µm × 10 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for nanoscale phosphoproteome profiling of mass-limited samples at the low nanogram level not accessible by current proteomics platforms.