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单细胞中单个氨基酸变异的发现。

Single Amino Acid Variant Discovery in Small Numbers of Cells.

机构信息

Department of Surgery , University of Michigan , Ann Arbor , Michigan 48109 , United States.

NCMIS, RCSDS, Academy of Mathematics and Systems Science , Chinese Academy of Sciences , Beijing 100190 , China.

出版信息

J Proteome Res. 2019 Jan 4;18(1):417-425. doi: 10.1021/acs.jproteome.8b00694. Epub 2018 Nov 21.

Abstract

We have performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing, and a carrier/reference strategy. Off line fractionation of the TMT-labeled sample pooled with TMT-labeled carrier Panc-1 whole cell proteome was achieved using alkaline reversed phase spin columns. The fractionation in conjunction with the carrier/reference (C/R) proteome allowed us to detect 47 414 unique peptides derived from 6261 proteins, which provided a sufficient coverage to search for single amino acid variants (SAAVs) related to cancer. This high sample coverage is essential in order to detect a significant number of SAAVs. In order to verify genuine SAAVs versus false SAAVs, we used the SAVControl pipeline and found a total of 79 SAAVs from the 9-cell Panc-1 sample and 174 SAAVs from the 5000-cell Panc-1 C/R proteome. The SAAVs as sorted into high confidence and low confidence SAAVs were checked manually. All the high confidence SAAVs were found to be genuine SAAVs, while half of the low confidence SAAVs were found to be false SAAVs mainly related to PTMs. We identified several cancer-related SAAVs including KRAS, which is an important oncoprotein in pancreatic cancer. In addition, we were able to detect sites involved in loss or gain of glycosylation due to the enhanced coverage available in these experiments where we can detect both sites of loss and gain of glycosylation.

摘要

我们使用样品分馏、TMT 多重标记和载体/参考策略,对少至 9 个 Panc-1 细胞进行了深度蛋白质组学分析。使用碱性反相旋转柱对 TMT 标记的样品与 TMT 标记的载体 Panc-1 全细胞蛋白质组混合液进行离线分馏。分馏与载体/参考(C/R)蛋白质组相结合,使我们能够检测到 47414 个独特的肽段,来自 6261 种蛋白质,这为寻找与癌症相关的单个氨基酸变异(SAAV)提供了足够的覆盖度。这种高样品覆盖率对于检测大量 SAAV 至关重要。为了验证真正的 SAAV 与假 SAAV,我们使用了 SAVControl 管道,从 9 个细胞的 Panc-1 样本中总共发现了 79 个 SAAV,从 5000 个细胞的 Panc-1 C/R 蛋白质组中发现了 174 个 SAAV。SAAV 被分类为高可信度和低可信度 SAAV,然后手动检查。所有高可信度的 SAAV 都被证实是真正的 SAAV,而一半的低可信度 SAAV 被发现是假 SAAV,主要与 PTM 有关。我们鉴定了几个与癌症相关的 SAAV,包括 KRAS,它是胰腺癌中的一种重要癌蛋白。此外,我们还能够检测到由于这些实验中提供的增强覆盖度而导致的糖基化丢失或获得的位点,在这些实验中,我们可以检测到糖基化的丢失和获得位点。

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