Improved surfactant-assisted one-pot sample preparation for robust convenient single cell proteomics

Abstract

Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field for comprehensive characterization of cellular heterogeneity. However, most of the current SCP approaches employ sub-{micro}L to 1 {micro}L processing volume for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to the plate well to compensate water evaporation, which greatly limits their broad accessibility and analytical robustness. Here we report a robust convenient SCP method termed iSOP (improved Surfactant-assisted One-Pot processing) for processing of single cells at the low {micro}L processing volume using the 384-well plate with tight sealing to avoid sample drying loss. This iSOP SCP method was built upon our previously developed SOP method by the replacement of a PCR tube or 96-well plate with the low-volume 384-well plate and systematic optimization of the single-cell processing conditions. After optimization, 3 {micro}L was selected as the processing volume with a mixture of 2 ng trypsin and 2 ng Lys-C enzymes in terms of robustness, detection sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify [~]1,200-1,800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines has demonstrated that iSOP-MS enabled reliable identification of an average of [~]1,700 and [~]2,050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity between the two distinct cell types and within the same cell type. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective quantitative SCP analysis.