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 workflows can be labor intensive and lead to long sample-to-answer times. Here we report a sample processing method that achieves cell lysis, protein denaturation and digestion in one hour with a single reagent dispensing step using commercially available high-temperature-stabilized proteases. Four different one-step reagent compositions were evaluated, and the mixture providing greatest proteome coverage was compared to the previously employed multi-step workflow. The one-step preparation increases proteome coverage relative to the multi-step 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 substrates, and found the polypropylene provided improved proteome coverage. Combined, the one-step sample preparation and the polypropylene substrates enabled identification of an average of nearly 2,400 proteins per cell using a standard data-dependent workflow with an Orbitrap mass spectrometer. As such, these advances significantly simplify sample preparation for single-cell proteomics and broaden accessibility with no compromise in terms of proteome coverage.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Ryan Kelly  

LAB HEAD: Dr Ryan T. Kelly

PROVIDER: PXD041879 | Pride | 2023-10-09

REPOSITORIES: Pride