A streamlined workflow for nanoscale phosphoproteome analysis

Effective nanoscale phosphoproteome analysis remains a daunting task, primarily due to significant sample losses associated with non-specific surface adsorption during phosphopeptide enrichment as well as other sample processing steps. To address this issue, we have developed a tandem tip-based C18-IMAC-C18 method which greatly reduces not only sample loss and sample transfer steps but also processing time for phosphopeptide enrichment (<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 approaches) for enhanced detection sensitivity and sample throughput leads to form a streamlined tandem tip-based workflow for enabling rapid sensitive nanoscale phosphoproteomics. This streamlined workflow allows for precise quantification of ~600 phosphopeptides from 100 MCF10A cells sorted by FACS. The abundance of the quantified phosphopeptides enabled for robust separation of EGF treated MCF10A cells from nontreated MCF10A cells. This workflow may open 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.