On-capillary Cell Lysis Enables Top-down Proteomic Analysis of Single Mammalian Cells by CE-MS/MS

Abstract

Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (∼1000 cells) and demonstrated 0−9 intact cells injected with a dependency on the duration of spray voltage application. Spray voltage applied for 2 min injected an average of 7 ± 2 cells and resulted in 33−57 protein and 40−88 proteoform identifications (N = 4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single HeLa cells (N = 4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17−40 proteins and 23−50 proteoforms identified. An additional cell line, THP-1, was analyzed at the single-cell level, and proteoform abundances were compared to show the capabilities of single-cell TDP (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.

Nanocapillary sampling coupled to liquid chromatography mass spectrometry delivers single cell drug measurement and lipid fingerprints

“…Single cell mass spectrometry has been the subject of a steadily growing number of publications in recent years. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] This is because bulk measurements of populations of cells does not identify heterogeneity, which is a fundamental property of all biological systems and has wide ranging consequences including for cell to cell communication, treatment of infectious diseases and cancers. 1 Therefore, analysing single cells is crucial to answering fundamental biological questions and has wide reaching impact including in drug discovery applications.…”

“…21 Recently, nanocapillary sampling approaches have been used to extract living cells or intra-cellular contents from 2D culture, which are then analysed using mass sprectrometry. 9,[22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] These approaches, also termed Video-MS, Live Single-Cell MS, or Direct Analyte Probe Nanoextraction (DAPNe) allow a target cell of interest to be extracted into a capillary under video-microscope observation. Nanocapillary sampling approaches can sample single, live cells whilst retaining spatial information and are therefore advantageous.…”

“…11 For example, developments in single cell proteomics has revealed heterogeneity in cellular protein signatures at an unprecedented level of detail. 9,[12][13][14] However, mass spectrometry analysis of smaller molecules at the single cell level presents a significant analytical challenge, due to the minute volume of cell content and the wide range of concentrations of metabolites, lipids and proteins in a cell. 15 The imaging mass spectrometry techniques, matrix assisted laser desorption/ionisation (MALDI) and secondary ionisation mass spectrometry (SIMS), have sufficient spatial resolution to detect metabolites and drugs in single eukaryotic cells.…”

“…11 For example, developments in single cell proteomics has revealed heterogeneity in cellular protein signatures at an unprecedented level of detail. 9,12–14 However, mass spectrometry analysis of smaller molecules at the single cell level presents a significant analytical challenge, due to the minute volume of cell content and the wide range of concentrations of metabolites, lipids and proteins in a cell. 15…”

High Sensitivity Top-down Proteomics Captures Single Muscle Cell Heterogeneity in Large Proteoforms

“…Although a previous study by Zhu et al has used the nanoPOTs platform for the extraction and sampling of intact proteins from low numbers of HeLa cells, only small proteoforms (<15 kDa) were detected (69). Recently, a capillary electrophoresis coupled to MS/MS (CE-MS/MS) approach has been used for analysis of proteins from single-cells, nonetheless, the detected proteoforms were almost exclusively below 15 kDa with no proteins greater than 30 kDa (70). Top-down MS analysis of large proteins is challenging due to an exponential decay in S/N with increasing molecular weight (MW) as well as co-elution with low MW and high abundance proteins in a mixture (29, 33, 65).…”