Authors
Reta Birhanu Kitata, Zhangyang Xu, Nadia Bayou, Rui Zhao, William B Chrisler, Matthew J Gaffrey, Karl K Weitz, Mara Serena Serafini, Elisabetta Molteni, Vladislav A Petyuk, Massimo Cristofanilli, Tao Liu, Carolina Reduzzi, Tujin Shi
Published in
Journal of proteome research. Jul 10, 2026. Epub Jul 10, 2026.
Abstract
Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field. However, current SCP approaches generally employ sub-μL to 1 μL processing volumes for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to compensate for evaporation, which limits their broad accessibility and analytical robustness. Here, we report a robust and convenient SCP method termed iSOP (improved surfactant-assisted one-pot processing) for the processing of single cells at a low μL processing volume using a 384-well plate with tight sealing to avoid sample drying loss. After systematic optimization, 3 μL was selected for iSOP as the processing volume, with a mixture of trypsin and Lys-C enzymes (2 ng of each enzyme) in terms of robustness, sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify ∼1200-1800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines enabled reliable identification of an average of ∼1700 and ∼2050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective, quantitative SCP analysis.
PMID:
42430151
Bibliographic data and abstract were imported from PubMed on 10 Jul 2026.
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