Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling

Abstract

In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 μm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 μm i.d. nano-LC packing column with 3 μm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7–170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics

“…The performance of SOP-MS (e.g., sensitivity, reproducibility, and quantitation accuracy) was demonstrated by label-free MS analysis of low mass inputs from a serial dilution of uniform MCF7 cell lysates, LCMdissected small tissue sections, and FACS-sorted single cells. Based on the actual MS/MS spectra for reliable protein identification (without using the MBR function) which is the cornerstone of MS-based proteomics, SOP-MS can identify~146 protein groups from single human cells, higher than~128 for iPAD1-MS 24 and 51 for OAD-MS 25 and~1.4-2.5-fold lower than~211-362 for nanoPOTS-MS [55][56][57] (Supplementary Table 1), and~1200 proteins from small tissue sections (close to~20 cells). Comparative analysis of single MCF10A cells using both SOP-MS and nanoPOTS-MS has shown that the number of protein groups from SOP-MS is~1.6-fold lower than that from nanoPOTS-MS and~60% of protein groups from SOP-MS overlapped with the protein groups from nanoPOTS-MS ( Supplementary Fig.…”

“…However, single-cell proteomic analysis of regular-size mammalian cells (typically~100 pg per cell) remains highly challenging, primarily due to technical difficulties in effective sampling and processing [21][22][23] . In recent three years great progress has been made to improve processing recovery from low numbers of cells by either reducing sample processing volume (e.g., nanoPOTS, OAD, and iPAD-1 devices downscaling the processing volume to~2-200 nL for label-free global proteomics 21,24,25 ) or using excessive amounts of carrier proteins or proteome (e.g., the addition of exogenous BSA as a carrier protein for targeted proteomics 22,23 or tandem mass tag (TMT)labeled 100s of cells as a carrier channel for TMT labeling-based global proteomics 26 ). However, all these approaches have technical drawbacks: nanoPOTS, OAD, and iPAD-1 are not easily adoptable for broad benchtop applications 21,24,25 ; exogenous protein carrier is more suitable for targeted proteomics.…”

“…In recent three years great progress has been made to improve processing recovery from low numbers of cells by either reducing sample processing volume (e.g., nanoPOTS, OAD, and iPAD-1 devices downscaling the processing volume to~2-200 nL for label-free global proteomics 21,24,25 ) or using excessive amounts of carrier proteins or proteome (e.g., the addition of exogenous BSA as a carrier protein for targeted proteomics 22,23 or tandem mass tag (TMT)labeled 100s of cells as a carrier channel for TMT labeling-based global proteomics 26 ). However, all these approaches have technical drawbacks: nanoPOTS, OAD, and iPAD-1 are not easily adoptable for broad benchtop applications 21,24,25 ; exogenous protein carrier is more suitable for targeted proteomics. Peptides from excessive exogenous proteins are frequently sequenced by MS/MS, which greatly reduces the chance for sequencing low abundant endogenous peptides 22,23 ; a TMT carrier is added after sample processing, and thus it cannot effectively prevent the surface adsorption losses during initial sample processing 26 , resulting in low reproducibility with a correlation coefficient of only~0.2-0.4 between replicates for ineffectively processed single cells 27 .…”

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

“…they are either label free or use isobaric labeling. In the label-free methods 5-10 , single cells are individually processed and analyzed using liquid chromatography-mass spectrometry (LC-MS) signal intensity measurements (i.e. MS1 ion currents) to quantify protein abundance.…”

DO-MS: Data-Driven Optimization of Mass Spectrometry Methods

“…[1][2][3][4] Advances in LC-MS/MS have enabeled analysis of protein complexes and their functions, [5][6][7][8][9] regulation of protein synthesis and alternative RNA translation, 10,11 rare cells in blood, 12,13 and protein conformations. [14][15][16] The increasing sensitivity, [17][18][19][20][21][22][23] throughput, and robustness 24 of LC-MS/MS set the stage for quantifying thousands of proteins across many thousands of single cells, providing data with transformative potential for biomedical research. [25][26][27][28][29] While LC-MS/MS proteomics methods are very powerful, they require extensive optimization of interdependent instrument parameters.…”