Fully automated sample processing and analysis workflow for low-input proteome profiling

Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples for characterization of cellular heterogeneity and high-resolution mapping of tissues. Several techniques used to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited the implementation in most proteomics laboratories. Here we developed a fully automated platform termed autoPOTS (Automated Preparation in One pot for Trace Samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was evaluated and utilized for fully automated sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30-µm-i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a modest reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. As a test of clinical feasibility, we used autoPOTS to identify an average of 1070 protein groups from ~130 fluorescence-activated cell sorted (FACS) B or T lymphocytes. The dataset here includes all the raw files of HeLa cells and lymphocytes. We anticipate that the simplicity and ease of implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.