Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC‐MS

Abstract

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells

“…Using nanoPOTS in combination with ultrasensitive LC-MS/MS, >3000 protein groups have been identified from as few as 10 mammalian cells, which is a depth of proteome coverage not previously achieved for fewer than 1000 cells. nanoPOTS has also been used to identify ∼700 proteins from single mammalian cells 26. While nanoPOTS provides an unprecedented depth of proteome coverage at and near the single cell level, it will be challenging to substantially increase proteome coverage without additional breakthroughs in sensitivity due to the use of a 1D nanoLC separation.…”

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

“…Using nanoliter-scale oil air droplet (OAD) chip, single HeLa cells and mouse oocytes were prepared for bottom-up analysis on an orbitrap instrument leading to the identification of~40 to~250 proteins on average, respectively [113]. Zhu et al further downscaled and partially automated the process for proteomic sample handling with the development of the nanodroplet processing in one-pot for trace proteomic samples (nanoPOTs) ( Figure 6) [88,114]. The NanoPOTs device consists of microfabricated glass chips with photolithographed hydrophilic pedestals (Figure 6ab).…”

Proteome analysis of tissues by mass spectrometry

“…Further, proteomic analysis of glioblastoma stem‐like cells (GSC) which are thought to have significant role in tumor recurrence, identified antigens that induce tumor‐specific T cell responses and might be important targets in future therapies (Rapp et al, ). Moreover, cell sorting was further downstreamed to single cell proteomics by combination of fluorescence‐activated cell sorting (FACS) and nanoPOTS platform whereas 485 proteins were identified across the single‐cell samples (Y Zhu, Clair, et al, ).…”