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An integrated proteome analysis device for fast single cell protein profiling Xi Shao, Xuantang Wang, Sheng Guan, Haizhu Lin, Guoquan Yan, Mingxia Gao, Chun-hui Deng, and Xiangmin Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03692 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018
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Analytical Chemistry
An integrated proteome analysis device for fast single cell protein profiling Xi Shao, Xuantang Wang, Sheng Guan, Haizhu Lin, Guoquan Yan, Mingxia Gao, Chunhui Deng, Xiangmin Zhang* Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China
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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ABSTRACT: In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteome from 100 cells.1 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 hour. In the iPAD-1, 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 LC column directly through a true zero dead volume union, to minimize protein transferring loss. A home-made 22-μm-i.d. nanoLC packing column with 3-μm-i.d. ESI-tip was used in the device to achieve an 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 hour. In analysis of ten single HeLa cells, maximum 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS, and the detection limit was estimated around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was ~120 fold higher than our previous developed iPAD-100 system1. Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of cell-cycle of tested HeLa cells by amount of core histone proteins.
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Analytical Chemistry
Nowadays, single cell analysis has attracted plenty of attention in scientific community.26
Since cellular heterogeneity is a widespread phenomenon even within the same
population,6 it is important to know individual information of each cell. An average value in analysis of a sample containing a large number of cells cannot reveal remarkable features of single cell.7 Therefore, methods of single cell analysis must be developed. Single cell analysis could provide specific information of cellular heterogeneity,8 which is powerful to distinguish cell phenotypes and functions in cell biology.9,10 It could also obtain accurate quantitative information of a cell by counting molecular copy numbers.11,12 Additionally, single cell analysis could offer unique information in stem-cell differentiation,13 cellcycle,14 rare cells like circulating tumor cells (CTCs),15 and so on. Single cell proteome analysis is a great challenge mainly because protein content in single cell was rather low, while proteins couldn’t be amplified like genes. Usually, the diameter of a mammalian cell is ranged from 10 to 20 μm;6 the total protein amount is below 16 fmol (0.4 ng);16 and the median protein copy number is only 50,000 (~80 zmol) among many kinds of proteins in a single cell.17 In recent years, single mammalian cell proteome analysis based on mass cytometry,18 microfluidic chip,19,20 Western blotting21,22 and so on were reported. The methods are fairly useful in target protein detection, but the number of identified proteins are likely to reach a limit of around 50 due to their antibodyreliance in detection.23 As we know, nano-LC-MS/MS is widely used in proteome analysis. It is also one of the most potential techniques in realizing hundreds to thousands of protein identification from single mammalian cell.2,3 Treatments of small amount of mammalian cells (
