on 06-19-202003:25 PM - edited on 10-15-202110:52 AM by AnalyteGuru
Khatereh Motamedchaboki1; Maowei Dou1; Yongzheng Cong2; Romain Huguet1; Aaron M Robitaille1; David M Horn1; Yufeng Shen3; Daniel Lopez-Ferrer1; Ryan T Kelly2, 4; Ying Zhu4. ASMS 2020 Traditionally, proteomics experiments are applied to large populations of cells, representing the average protein expression under given biological conditions. However, understanding the cellular heterogeneity provides insights that cannot be gained from bulk studies, such that the analysis of single-cell protein expression is of growing interest. Current LC-MS-based proteomics workflows have not been widely applicable to single cell analysis, mainly due to large sample losses during sample preparation, limited analytical sensitivity and low throughput. To address these challenges, we have combined nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) technology with tandem mass tag (TMT) isobaric labeling to analyze single mammalian cells containing ~0.2 ng total proteins on the Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer with real time search and the Thermo Scientific™ FAIMS Pro™ Interface to improve single cell proteome coverage and enhance quantitation accuracy. The FAIMS-enhanced label-free workflow resulted in the identification of an average of 829 protein groups from single Hela cells with high-confidence MS2 spectra. This was 3-fold higher than without FAIMS. The Thermo Scientific™TMT10plex™ analysis of three cultured murine cell populations were compared with MS2 and SPS MS3 method with Real Time Search. We have demonstrated that single cell proteomes can be quantified using label-free or TMT workflows by combing nanoPOTS with the Orbitrap Eclipse Tribrid mass spectrometer, and the FAIMS Pro Interface, enabling researchers to investigate cell heterogeneity as well as rare cells in an ultra-sensitive, higher throughput LC-MS analysis.
1Thermo Fisher Scientific, San Jose, California; 2Brigham Young University, Provo, UT; 3CoAnn Technologies LLC, Richland, WA; 4Pacific Northwest National Laboratory, Richland, WA