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.

Easy and Accessible Workflow for Label-Free Single-Cell Proteomics

“…Each well contained manually pipetted buffer solution, and the dispensed sodium fluorescein solutions were calculated to produce a final volume and concentration in each well of 20 μL and 6 μM, respectively. To approximate the viscosity and surface-wetting properties of our one-step sample preparation reagent solution, all solutions contained 0.05% of the nonionic surfactant DDM. Given the low concentration of DDM, we evaluated two classes of reagent dispensing cassettes: those that require an added surfactant (RS) and those designed to work with surfactant-free (SF) solutions.…”

“…The Rapid-Digestion Trypsin/Lys-C kit (Part No. VA1061, Promega, Madison, WI) with 0.05% DDM (Sigma-Aldrich) served as the reagent mixture to accomplish cell lysis, protein denaturation, and digestion in a single step as described previously . This one-step sample preparation reagent was dispensed using the HP D100 Single Cell Dispenser with the requiring-surfactant cassette and with the fluid class “Aqueous + Brij 35”.…”

“…Here, we utilize the newly released D100 Single Cell Dispenser from HP Inc. to isolate and prepare single cells for proteome profiling. In combination with our recently developed one-step sample preparation cocktail and standard 384-well PCR plates, the HP D100 provides an easy, low-cost, fully commercial, and widely accessible workflow for SCP sample preparation. We characterized the single-cell and reagent dispensing performance of the HP D100 and evaluated proteome coverage for different sample preparation volumes and protease concentrations.…”

“…However, these both reduced proteome coverage relative to nanoPOTS and were thus better suited to samples comprising tens or hundreds of cells. Efforts have also been made to simplify sample preparation by reducing the number of required steps, particularly for label-free SCP, ,− although these have required custom substrates and costly dispensing instrumentation and/or resulted in reduced proteome coverage.…”

Toward Single Bacterium Proteomics

“…The key innovation in SCoPE MS was the use of the carrier proteome (CP) composed of 100–200 cells and multiplexed with single cell samples using isobaric tandem mass tag (TMT). With SCoPE-MS, it is habitually possible to analyze human cells to the depth of 1500–2000 proteins. , There are dozens of recent reports with similar or greater proteome analysis depths owing to continuous advances in sample preparation, − data acquisition expanding the choice of analytical approaches to label-free technique and with data independent acquisition modes, ,− as well as providing optimal data analysis. , …”