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

ABSTRACT: Recent advances in mass spectrometry-based single-cell proteomics (SCP) have revolutionized the SCP field for comprehensive characterization of cellular heterogeneity. However, current SCP approaches generally employ sub-µL to 1 µL sample processing volume using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to compensate evaporation, which greatly limits their broad accessibility and robustness. Here we report a robust convenient SCP method termed iSOP (improved Surfactant-assisted One-Pot processing) for processing of single cells at low µL volume using 384-well plate with tight sealing to avoid sample drying loss. This iSOP SCP method was built upon our previously developed SOP method by systematic optimization of processing conditions in a low-volume 384-well plate. After optimization, 3 µL was selected as processing volume with a mixture of trypsin and Lys-C enzymes (2 ng for each). With a commonly accessible LC-MS platform, iSOP-MS quantified ~1,200–1,800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines has 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. Overall, iSOP-MS provides a robust and convenient platform for routine, cost-effective, quantitative SCP analysis.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tujin Shi and Reta Birhanu Kitata 

PROVIDER: PXD072373 | JPOST Repository | Sat May 30 00:00:00 BST 2026

REPOSITORIES: jPOST