Fully Automated Sample Processing and Analysis Workflow for Low-Input Proteome Profiling

Abstract

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.

Figure 1.

Workflow for automated sample processing and analysis of low-input samples. (a) Cells are dispensed into prefilled wells of a 384-well plate using any of the three sample isolation methods: (1) limiting dilution from cell suspension, (2) FACS, or (3) capillary-based micromanipulation. (b) After collecting cells, wells are sealed with a prescored silicone cover and placed on the OT-2 temperature control module. Cell lysis, protein reduction, alkylation, and digestion are performed in a one-pot workflow; buffer or water is added periodically to compensate for limited evaporation. (c) The well plate is loaded onto an autosampler for nanoLC-MS/MS analysis.

Figure 2.

AutoPOTS sample preparation workflow. Python protocols 1 and 2 are performed in the OT-2, while cell isolation is performed separately.

Figure 3.

Modified autosampler for low-input sample injection and analysis. A 10-port nanovolume valve replaces the standard 6-port valve and is connected a 30 μm i.d. analytical column, an SPE column, a split-flow column, a 7 μL sample loop, an injection needle, and the autosampler syringe. (a) Sample is loaded into the sample loop. (b) The valve is switched to the injection position and flow from the split column drives the sample from the sample loop to the SPE column. (c) The valve is switched back to the load position and the sample is separated by gradient-elution reversed-phase nanoLC at a flow rate of ~40 nL/min.

Figure 4.

Number of peptides (a) and proteins (b) identified by MS/MS from 1, 10, ~150, and ~500 HeLa cells. (c) Overlap of protein groups identified in two of three replicates from various sample loadings.

Figure 5.

Proteome profiling of ~130 human B and T lymphocytes. (a) Spearman correlations of each sample. (b) Significant proteins (orange) identified using the t-test (p-value > 0.01) from the 961 protein groups identified in at least three of five replicates for each cell type.