Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.
Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.
复制标题
DOI：
10.1073/pnas.1721899115
复制
发表时间：
2018-03-13
影响因子：
11.1
通讯作者：
Wells JA
中科院分区：
文献类型：
--
作者：
Pollock SB;Hu A;Mou Y;Martinko AJ;Julien O;Hornsby M;Ploder L;Adams JJ;Geng H;Müschen M;Sidhu SS;Moffat J;Wells JA
下一代测序（NGS）允许对基因组和转录组进行全面研究。然而，一个类似的广泛的，高度复用的，和廉价的方法，蛋白质组学使用NGS仍然难以捉摸。在这里，我们描述了一种基于噬菌体展示的方法，该方法使用预先选择的抗体，这些抗体是遗传编码的，并且能够以低成本同时分析培养物中细胞上的数百个细胞表面靶标或单独分析，而不需要与纯化的抗体进行化学缀合。我们使用这种方法来识别癌细胞中发生变化的细胞表面蛋白，其中一些蛋白受到协调调节，可能导致新的生物标志物和癌症靶点。人类细胞表达数千种不同的表面蛋白，可用于细胞分类或区分健康和疾病状况。一种能够同时且廉价地对表面蛋白质组的相当大一部分进行分析的方法将使得能够对细胞状态进行更准确和完整的分类。我们提出了一种高度多重和定量的表面蛋白质组学方法，使用基因条形码抗体称为噬菌体抗体下一代测序（PhaNGS）。使用144个预选抗体展示在丝状噬菌体（Fab噬菌体）对44个受体的目标，我们评估B细胞表面蛋白的变化后，急性淋巴细胞白血病（ALL）患者的耐药性的发展和适应癌基因表达的Myc诱导的伯基特淋巴瘤模型。我们进一步表明PhaNGS可以应用于单细胞水平。我们的研究结果表明，一组共同的蛋白质，包括FLT 3，NCR 3LG 1，和ROR 1占主导地位的B细胞类似的致癌扰动的反应。将高亲和力、选择性、遗传编码的结合剂与NGS连接能够实现直接和高度多重的蛋白质检测，与mRNA的RNA测序相当。PhaNGS具有同时且廉价地分析表面蛋白质组的相当大部分的潜力，从而能够对细胞状态进行更准确和完整的分类。
Next-generation sequencing (NGS) has allowed the comprehensive study of the genome and transcriptome. However, a similarly broad, highly multiplexed, and inexpensive method for proteomics using NGS remains elusive. Here, we describe a phage display-based method using preselected antibodies that are genetically encoded and capable of simultaneous profiling of hundreds of cell-surface targets on cells in culture or singly at low cost and without the need for chemical conjugation to purified antibodies. We use the method to identify cell-surface proteins that change in cancer cells, some of which are coordinately regulated and could lead to new biomarkers and cancer targets. Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states.
登录
查看更多内容
DOI：
10.1126/science.1198704
发表时间：
2011-05-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Bendall SC;Simonds EF;Qiu P;Amir el-AD;Krutzik PO;Finck R;Bruggner RV;Melamed R;Trejo A;Ornatsky OI;Balderas RS;Plevritis SK;Sachs K;Pe'er D;Tanner SD;Nolan GP
通讯作者：
Nolan GP
影响因子：
5.6
作者：
Ewert, S;Huber, T;Plückthun, A
通讯作者：
Plückthun, A
影响因子：
5.6
作者：
Persson, Helena;Ye, Wei;Wernimont, Amy;Adams, Jarrett J.;Koide, Akiko;Koide, Shohei;Lam, Robert;Sidhu, Sachdev S.
通讯作者：
Sidhu, Sachdev S.
DOI：
10.1074/mcp.o115.052209
发表时间：
2015-10
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Hornsby M;Paduch M;Miersch S;Sääf A;Matsuguchi T;Lee B;Wypisniak K;Doak A;King D;Usatyuk S;Perry K;Lu V;Thomas W;Luke J;Goodman J;Hoey RJ;Lai D;Griffin C;Li Z;Vizeacoumar FJ;Dong D;Campbell E;Anderson S;Zhong N;Gräslund S;Koide S;Moffat J;Sidhu S;Kossiakoff A;Wells J
通讯作者：
Wells J
影响因子：
11.4
作者：
Daneshmanesh, A. H.;Hojjat-Farsangi, M.;Mellstedt, H.
通讯作者：
Mellstedt, H.