Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics

Abstract

Sample preparation for single-cell proteomics is generally performed in a one-pot workflow with multiple dispensing and incubation steps. These hours-long processes can be labor intensive and lead to long sample-to-answer times. Here we report a sample preparation method that achieves cell lysis, protein denaturation, and digestion in 1 h using commercially available high-temperature-stabilized proteases with a single reagent dispensing step. Four different one-step reagent compositions were evaluated, and the mixture providing the highest proteome coverage was compared to the previously employed multistep workflow. The one-step preparation increases proteome coverage relative to the previous multistep workflow while minimizing labor input and the possibility of human error. We also compared sample recovery between previously used microfabricated glass nanowell chips and injection-molded polypropylene chips and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled the identification of an average of nearly 2400 proteins per cell using a standard data-dependent workflow with Orbitrap mass spectrometers. These advances greatly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.

Figure 1.

Evaluation of four one-step sample preparation mixtures in the analysis of single HeLa cells. (a) Peptide coverage. (b) Proteome coverage. (c) Peptide hydrophobicity as measured by GRAVY score. Error bars are ±1 standard deviation for n = 12 replicates.

Figure 2.

Comparison of the Protease-Only and Protease + DDM one-step mixtures. (a) Pearson correlation. (b). Venn diagram of quantifiable proteins.

Figure 3.

Comparison of one-step and multistep sample processing methods. (a) Proteome coverage (n = 10). (b) Pearson correlation between the one-step and multistep methods. (c) Comparison of GRAVY scores between the two methods.

Figure 4.

Evaluation of glass and polypropylene substrates for nanowell sample preparation. (a) Photographs of glass and polypropylene chips. Nanowells in the glass substrates were filled with 200 nL of DMSO for visualization. Peptide and proteome coverages are shown in (b) and (c), respectively (n = 10).