van der Mijn Johannes C, Labots Mariette, Piersma Sander R, Pham Thang V, Knol Jaco C, Broxterman Henk J, Verheul Henk M, Jiménez Connie R
Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
J Proteomics. 2015 Sep 8;127(Pt B):259-63. doi: 10.1016/j.jprot.2015.04.006. Epub 2015 Apr 16.
Mass spectrometry based phosphoproteomics emerged as advantageous approach for the analysis of tyrosine phosphorylation on proteins and tyrosine kinase signaling. Immunoaffinity purification is required for comprehensive analysis. Here we compared the performance of two antibodies for label-free phosphotyrosine-based phosphoproteomics.
Phosphopeptide immunoprecipitation of six technical replicates corresponding to 10mg protein from HCT116 cells was performed using agarose bead-coupled phosphotyrosine antibodies P-Tyr-1000 (N=3) and 4G10 (N=3). NanoLC-MS/MS was performed using a Q Exactive mass spectrometer. For relative quantitation of protein phosphorylation, spectral counts of phosphoproteins and ion intensities of phosphopeptides were determined using MaxQuant.
From the 3 samples incubated with P-Tyr-1000 a total of 689 phosphopeptides were identified with 60% ID reproducibility. The phosphopeptide capture using 4G10 resulted in a total of 421 at 46% ID reproducibility. The P-Tyr-1000 was applied to EGFR mutated U87 cells. Erlotinib reduced EGFR phosphorylation with 59% at y978, y1125, y1138, y1172, and y1197. EGFR inhibition was accompanied by enhanced phosphorylation of FYN, MET, PTK2, DYRK1A, MAPK1 and EPHA2.
The P-Tyr-1000 phosphotyrosine antibody performs superiorly when compared to 4G10 antibody for label-free phosphotyrosine-based phosphoproteomics. This workflow allows evaluation of drug target phosphorylation and may give insights in the pharmacodynamic effects of tyrosine kinase inhibitors.
In the past decade multiple tyrosine kinase inhibitors (TKIs) have been implemented in standard treatment regimens for patients with cancer. Unfortunately the majority of patients develops resistance to these drugs. Reliable tools for analysis of pharmacodynamic effects and drug resistance mechanisms are therefore warranted. Phosphoproteomic analyses have meanwhile emerged as a sophisticated approach for the determination of protein phosphorylation. These analyses rely on antibodies for enrichment of tyrosine-phosphorylated peptides. Here we compared two commercially available phosphotyrosine antibodies and show that P-Tyr-1000 yields 64% more phosphopeptides than 4G10 antibody, while including almost all 4G10 captured phosphopeptides. The workflow can be reproducibly performed at intermediate protein input levels of 10mg. Furthermore, application of the P-Tyr-1000 antibody in a standardized phosphoproteomics workflow allows relative quantitation of drug target inhibition and provides insights in alternative signaling pathways in cancer cells. This article is part of a Special Issue entitled: HUPO 2014.
基于质谱的磷酸化蛋白质组学已成为分析蛋白质酪氨酸磷酸化和酪氨酸激酶信号传导的优势方法。全面分析需要免疫亲和纯化。在此，我们比较了两种抗体用于基于无标记磷酸酪氨酸的磷酸化蛋白质组学的性能。
使用琼脂糖珠偶联的磷酸酪氨酸抗体P-Tyr-1000（N = 3）和4G10（N = 3）对来自HCT116细胞的相当于10mg蛋白质的六个技术重复进行磷酸肽免疫沉淀。使用Q Exactive质谱仪进行纳升液相色谱-串联质谱分析。为了对蛋白质磷酸化进行相对定量，使用MaxQuant确定磷酸化蛋白质的光谱计数和磷酸肽的离子强度。
在与P-Tyr-1000孵育的3个样品中，共鉴定出689个磷酸肽，鉴定重复性为60％。使用4G10进行磷酸肽捕获，共得到421个，鉴定重复性为46％。将P-Tyr-1000应用于EGFR突变的U87细胞。厄洛替尼使EGFR在y978、y1125、y1138、y1172和y1197处的磷酸化降低了59％。EGFR抑制伴随着FYN、MET、PTK2、DYRK1A、MAPK1和EPHA2磷酸化的增强。
与4G10抗体相比，P-Tyr-1000磷酸酪氨酸抗体在基于无标记磷酸酪氨酸的磷酸化蛋白质组学中表现更优。此工作流程可评估药物靶点磷酸化，并可能提供酪氨酸激酶抑制剂药效学效应的见解。
在过去十年中，多种酪氨酸激酶抑制剂（TKIs）已被纳入癌症患者的标准治疗方案。不幸的是，大多数患者会对这些药物产生耐药性。因此，需要可靠的工具来分析药效学效应和耐药机制。与此同时，磷酸化蛋白质组学分析已成为测定蛋白质磷酸化的一种精密方法。这些分析依赖于抗体来富集酪氨酸磷酸化肽段。在此，我们比较了两种市售的磷酸酪氨酸抗体，结果表明P-Tyr-1000产生的磷酸肽比4G10抗体多64％，同时几乎包含了4G10捕获的所有磷酸肽。该工作流程可在10mg的中等蛋白质输入水平上重复进行。此外，将P-Tyr-1000抗体应用于标准化的磷酸化蛋白质组学工作流程中，可对药物靶点抑制进行相对定量，并提供癌细胞中替代信号通路的见解。本文是名为：HUPO 2014的特刊的一部分。
