A streamlined tandem tip-based workflow for sensitive nanoscale phosphoproteomics

Effective nanoscale phosphoproteome analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during phosphopeptide enrichment and other sample processing steps. To address this issue, we have developed a tandem tip-based C18-IMAC-C18 method which significantly reduces not only sample loss but also sample transfer steps and processing time for phosphopeptide enrichment (to <20 min). The tandem tip method enables identification of >3,000 and >9,500 phosphopeptides from 1 and 10 µg of cell lysate inputs using the label-free method without the spectral library, respectively. Integration of the tandem tip method with our recently developed SOP (Surfactant-assisted One-Pot sample preparation; for nanoscale processing) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling; for enhanced detection sensitivity and sample throughput) approaches into a streamlined tandem tip-based workflow enables rapid and sensitive nanoscale phosphoproteome measurements. This streamlined workflow allows for precise quantification of ~600 phosphopeptides from 100 MCF10A cells sorted by FACS, and robust separation of EGF-treated and nontreated MCF10A cells. This workflow opens new avenues for nanoscale phosphoproteome profiling of small numbers of cells and mass-limited samples at the low nanogram levels, which cannot be accessed by current proteomics platforms.