Overall workflows and numbers of proteins identified from various methods. Source: Mun et al., 2024. “Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry”, Commun Biol 7, 884 (2024). Licensed under a Creative Commons Attribution 4.0 International License.

Demonstrating significant advancements in single-cell proteomics, Mun et al’s research enabled unbiased measurements of proteomes and post-translational modifications (PTMs) at the single-cell and single organelle-level.

The researchers employed a data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) approach to analyze single cells from normal cholangiocytes and cholangiocarcinoma cell lines. They identified an average of 2,548 proteins per single cell, totaling 4,197 proteins across 200 single cells.

Using network analysis, the study uncovered diversity in kinase expression within single cells, providing insights into signaling heterogeneity. The researchers successfully detected mutant proteins, including KRAS G12D, at the single-cell level.

The researchers extended their approach to single nuclei, demonstrating the potential to capture subcellular proteome dynamics and measure drug-induced epigenetic changes. They observed a reduction in trimethylation of H3K27 upon treatment with tazemetostat, an epigenetic inhibitor.

This study showcases the power of single-cell proteomics to elucidate cellular heterogeneity, detect mutant proteins, and measure PTMs at unprecedented resolution. These insights complement single-cell genomics data and offer a more comprehensive understanding of cellular states and drug responses.

The researchers used an IonOpticks Aurora Elite CSI 15×75 C18 UHPLC column for peptide separation, which contributed to the high-quality proteome coverage achieved in this study.

Publication
Communications Biology

Authors

Dong-Gi Mun, Firdous A. Bhat, Neha Joshi, Leticia Sandoval, Husheng Ding, Anu Jain, Jane A. Peterson, Taewook Kang, Ganesh P. Pujari, Jennifer L. Tomlinson, Rohit Budhraja, Roman M. Zenka, Nagarajan Kannan, Benjamin R. Kipp, Surendra Dasari, Alexandre Gaspar-Maia, Rory L. Smoot, Richard K. Kandasamy & Akhilesh Pandey

Title

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry