化学
蛋白质组学
色谱法
样品制备
质谱法
吞吐量
单细胞分析
多路复用
等压标记
定量蛋白质组学
样品(材料)
细胞
串联质谱法
计算机科学
生物信息学
生物化学
蛋白质质谱法
生物
基因
电信
无线
作者
Sarah Williams,Andrey Liyu,CF Tsai,Ronald J. Moore,Daniel J. Orton,William B. Chrisler,Matthew J. Gaffrey,Tao Liu,Richard Smith,Ryan Kelly,Ljiljana Paša‐Tolić,Ying Zhu
出处
期刊:Analytical Chemistry
[American Chemical Society]
日期:2020-07-08
卷期号:92 (15): 10588-10596
被引量:103
标识
DOI:10.1021/acs.analchem.0c01551
摘要
Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.
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