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.

Figure 1.

Overview of intact cell injection workflows and cell suspension droplets. (A) Schematic of spray voltage injection workflow. The separation capillary is prepared for cell lysis and CE-MS/MS analysis by filling in first with BGE and then with a plug of lysis buffer. The capillary inlet is positioned in cell suspension (500 nL) pipetted on a glass slide (the zoomed capillary inlet in the droplet is shown in the red-frame insert), and electrospray voltage is applied at the MS inlet, drawing cells into the capillary. (B) Schematic of manual injection of single cells with hydrodynamic loading using height difference between the capillary inlet and outlet to generate flow and draw in the cell. In the injection flow panel, a 75% FA plug, (I) a single cell, (II) and another 75% FA plug (III) were loaded in this order for single-cell injection and lysis. (C) Representative image cell suspension droplet used for single-cell loading.

Figure 2.

Assessment of spray voltage injection of intact cells. Microscope images at 10× magnification of cells inside the capillary from (A) 1 min (N = 3) and (B) 2 min injection (N = 3). Red arrows indicate individual cells loaded inside the capillary. (C) Fluorescent (top) and bright-field images (middle) of cells stained with acridine orange inside the capillary. Overlaid images are shown at the bottom. (D) Cells counted in the capillary with 1 and 2 min spray injections. Mean counts ± STDEV are displayed.

Figure 3.

Demonstrating single-cell injection and cell lysis inside the capillary. (A) Fluorescent (top) and bright-field (middle) images of a representative single HeLa cell injected into the capillary by hydrodynamic pressure. Overlaid images are shown at the bottom. (B) Time course of lysis a single cell inside the capillary by a single plug of 75% FA. (C) Zoomed in images of the cell lysing by 75% FA at different time points.

Figure 4.

Protein and proteoform identifications from single-cell and spray voltage injections analyzed by CE-MS/MS. (A) Boxplots show the range and median of protein and proteoform identifications for each injection mode tested, as well as blank sample buffer and suspension buffer alone (N = 4). Dots indicate values from individual samples. (B) Single HeLa cell protein and proteoform identifications plotted against measured cell diameters. Linear trendlines demonstrate the correlation between protein and proteoform identifications with cell size. (C) S-curve of protein copy numbers per cell from a previously reported deep proteomic study of HeLa, with proteins identified from a single HeLa cell TDP in this study mapped in red. Selected proteins of interest are labeled.

Figure 5.

Representative MS1 and MS2 spectra from confidently identified histone proteoforms with different PTMs. (A) MS1 spectrum for co-migrating histone 4 proteoforms with dimethyl (yellow) and butyryl (green) PTM assignments. The top insert shows the extracted peaks for each proteoform. (B) MS2 spectrum with mapped fragments and fragmentation coverage indicating dimethylated (+28 Da) histone 4, identified in TDP analysis of a single HeLa cell. Putative modified lysine residue indicated with a red box. (C) MS2 spectrum with mapped fragments and fragmentation coverage indicating butyrylated (+70 Da) histone 4 identified in TDP analysis of a single HeLa cell. Putative modified lysine residue indicated with a red box.

Figure 6.

Assessment of MBR and comparison of HeLa and THP-1 single cell proteoform identifications. (A) Protein and proteoform identifications with and without MBR are shown for each single HeLa cell analyzed. The average number of identification with and without MBR is also shown. (B) Proteoform overlap between HeLa single cells shown for identifications without and with MBR. (C) Proteoform overlap between HeLa (N = 4) and THP-1 (N = 3) cells without and with MBR. (D) Principal components PC1 vs PC2 and PC1 vs PC3 are shown for PCA analysis of 79 proteoform abundances determined by MBR between HeLa and THP-1 cells. (N = 7 data points). (E) Heat map of proteoforms identified in single THP-1 (N = 3) and HeLa (N = 4) cells. Unsupervised hierarchical clustering was performed based on Euclidean distance using average linkage. Proteoform peak abundances are log2 transformed. Missing values for PCA and heat map clustering were imputed as 10% of the minimum observed value.