Comprehensive Micro-SPE-Based Bottom-Up Proteomic Workflow for Sensitive Analysis of Limited Samples

Clinical and biological samples are often scarce and precious (e.g., rare cell isolates, microneedle tissue biopsies, small-volume liquid biopsies, and even single cells or organelles). Typical large-scale proteomic methods, where significantly higher protein amounts are analyzed, are not directly transferable to the analysis of limited samples due to their incompatibility with pg-, ng-, and low-μg-level protein sample amounts. Here, we report the on-microsolid-phase extraction tip (OmSET)-based sample preparation workflow for sensitive analysis of limited biological samples to address this challenge. The developed platform was successfully tested for the analysis of 100–10,000 typical mammalian cells and is scalable to allow for lower and larger protein amounts and more samples to be analyzed (i.e., higher throughput of analysis).