Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze ...low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report 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 utilized for one-pot 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 moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.
Single-cell proteomics (SCP) can provide information that is unattainable through either bulk-scale protein measurements or single-cell profiling of other omes. Maximizing proteome coverage often ...requires custom instrumentation, consumables, and reagents for sample processing and separations, which has limited the accessibility of SCP to a small number of specialized laboratories. Commercial platforms have become available for SCP cell isolation and sample preparation, but the high cost of these platforms and the technical expertise required for their operation place them out of reach of many interested laboratories. Here, we assessed the new HP D100 Single Cell Dispenser for label-free SCP. The low-cost instrument proved highly accurate and reproducible for dispensing reagents in the range from 200 nL to 2 μL. We used the HP D100 to isolate and prepare single cells for SCP within 384-well PCR plates. When the well plates were immediately centrifuged following cell dispensing and again after reagent dispensing, we found that ∼97% of wells that were identified in the instrument software as containing a single cell indeed provided the proteome coverage expected of a single cell. This commercial dispenser combined with one-step sample processing provides a very rapid and easy-to-use workflow for SCP with no reduction in proteome coverage relative to a nanowell-based workflow, and the commercial well plates also facilitate autosampling with unmodified instrumentation. Single-cell samples were analyzed using home-packed 30 μm i.d. nanoLC columns as well as commercially available 50 μm i.d. columns. The commercial columns resulted in ∼35% fewer identified proteins. However, combined with the well plate-based preparation platform, the presented workflow provides a fully commercial and relatively low-cost alternative for SCP sample preparation and separation, which should greatly broaden the accessibility of SCP to other laboratories.
Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories to cost-effectively preserve valuable specimens for later study. With the rapid growth of spatial proteomics, FFPE ...tissues can serve as a more accessible alternative to more commonly used frozen tissues. However, extracting proteins from FFPE tissues is challenging due to cross-links formed between proteins and formaldehyde. Here, we have adapted the nanoPOTS sample processing workflow, which was previously applied to single cells and fresh-frozen tissues, to profile protein expression from FFPE tissues. Following the optimization of extraction solvents, times, and temperatures, we identified an average of 1312 and 3184 high-confidence master proteins from 10 μm thick FFPE-preserved mouse liver tissue squares having lateral dimensions of 50 and 200 μm, respectively. The observed proteome coverage for FFPE tissues was on average 88% of that achieved for similar fresh-frozen tissues. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. This modified nanodroplet processing in one pot for trace samples (nanoPOTS) and fully automated processing in one pot for trace sample (autoPOTS) workflows provides the greatest coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues. Data are available via ProteomeXchange with identifier PXD029729.
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