IS-SCP: enhanced single-cell proteomics using an in situ simplified strategy†

Abstract

Advances in single-cell proteomics have enabled the investigation of the distinctive proteomic makeup of individual cells, significantly impacting biomedical research. However, most existing approaches involve complex sample preparation workflows and are sensitive to potential sample loss, which limits their applicability. In this paper, we reported an advanced workflow for easy-to-use single-cell proteome analysis using an in situ simplified strategy, named “in situ simplified single-cell proteomics (IS-SCP)”. This workflow was developed following a comprehensive evaluation of reagent mix, volume, and reaction conditions, notably including the utilization of a cleavable surfactant, n-decyl-disulfide-β-D-maltoside (DSSM). In comparison to previous workflows that require multiple steps in sample preparation, the IS-SCP workflow simplifies the single-cell proteome pretreatment to a single step of adding single-cell samples into a mixed reagent, which increases the repeatability and depth of single-cell proteome analysis. The IS-SCP workflow was applied to the proteomic analysis of single mammalian tumor cells, specifically HeLa and A549 cells, resulting in the quantification of an average of 3021 and 3289 protein groups, respectively. These results showed the potential of this workflow for investigating cellular heterogeneity at a deep single-cell level.