打破WB&ELISA的局限? 不用抗体也能做WB? 目标蛋白定量分析的新宠

在蛋白组学中，蛋白验证实验是十分关键的一步，而提到蛋白验证，首先想到的便是Western Blot。Western Blot的实验原理是基于抗原抗体反应，通过相应的抗原抗体反应，从而验证不同处理样本间的蛋白表达水平的变化。然而并不是所有的蛋白都能找到相应的商业化抗体，另一方面自行设计合成抗体之路又是费时费力。即使现有的抗体也面临着特异性、稳定性难以保障的瓶颈，而且通量低、成本高。因此，传统WB在多方面限制了差异蛋白的验证，限制了课题的进一步开展。
 

PRM技术介绍

随着质谱技术的飞速发展，基于质谱的蛋白质组学在灵敏度、精确度、分辨率方面都有了极大的提升。PRM（Parallel Reaction Monitoring，平行反应监测）质谱技术的出现，很好的解决了Western Blot技术的局限性，通量高，可同时验证几十个蛋白，因此大大降低了单蛋白验证的成本，且前处理后的样本直接上质谱检测，无需抗体，摆脱了抗体依赖，提高了验证的准确性和成功率。

SRM技术是一项经典的蛋白组学靶向技术，也是靶向蛋白组学的金标准。SRM技术首先锁定母离子和其子离子，后续依赖三重四级杆（QqQ）进行检测。而PRM技术则是更进一层，用高分辨率质量分析器Orbitrap替代了三重四极杆的第三个四极杆，从而可以在一个统一的高分辨率质量分析中平行检测所有目标产物离子参考文献[1]，能够将干扰信息和真实的信号进行区分，从而保证更好的分析选择性。
 

图1 | PRM数据采集原理图示

PRM技术优势

● 继承了SRM技术的高灵敏度和特异性，由于Orbitrap的高分辨率和精准度，其灵敏度和特异性比SRM技术更高。
● 仅需要母离子的信息，即可进行实验，无需像SRM技术那般需要母离子/子离子对信息。
● 抗干扰能力强，排除虚假母离子/子离子对信息带来的信息干扰。
● 无需抗体，对不存在商业化抗体的蛋白验证，大幅度降低实验成本和实验时间。
● 重现性好，可以在多次重复检测中保证结果的稳定性。
 

PRM技术应用

● 受到物种和抗体等原因限制的蛋白验证实验；
● 蛋白质组学（DIA、iTRAQ、TMT等）后的差异目标蛋白验证；
● 临床疾病标志物的验证；
● 植物抗逆性研究标志物的验证；
……
 

PRM实验流程概述

1.选择待验证的蛋白。可以通过shot gun鉴定信息、分子生物学实验、文献报道等方式获得蛋白信息。
2.选取合适的实验组和对照组样品，进行前处理实验和DDA检测。
3.根据DDA搜库结果，skyline软件建库信息挑选可靠的靶向肽段（根据蛋白unique肽段数决定，2-5条肽段最佳）。
4.按照一定比例在样品中添加内标肽段iRT_{参考文献 [21]，}进行PRM检测。
5. 检测结果分析，包括内标肽段的质控、样品重复性验证等。
 

鹿明生物PRM部分展示

图2 | iRT保留时间
 

图3 | iRT质量偏差

图4 | 目标肽段峰形图

参考文献

[1].Peterson, A.C., et al., Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Molecular & cellular proteomics : MCP, 2012. 11(11): p. 1475 - 1488.
[2].Ronsein, G.E., et al., Targeted Proteomics Identifies Paraoxonase/Arylesterase 1 (PON1) and Apolipoprotein Cs as Potential Risk Factors for Hypoalphalipoproteinemia in Diabetic Subjects Treated with Fenofibrate and Rosiglitazone. Molecular & cellular proteomics : MCP, 2016. 15(3): p. 1083 - 1093.
[3].Taumer, C., et al., Parallel reaction monitoring on a Q Exactive mass spectrometer increases reproducibility of phosphopeptide detection in bacterial phosphoproteomics measurements. Journal of Proteomics, 2018.
[4].Lawrence, R.T., et al., Plug-and-play analysis of the human phosphoproteome by targeted high-resolution mass spectrometry. Nature Methods, 2016. 13(5): p. 431-434.
[5].Tang, H., et al., Multiplexed Parallel Reaction Monitoring Targeting Histone Modifications on the QExactive Mass Spectrometer. Analytical Chemistry, 2014. 86(11): p. 5526-5534.
[6].Gallien, S., S.Y. Kim and B. Domon, Large-Scale Targeted Proteomics Using Internal Standard Triggered-Parallel Reaction Monitoring (IS-PRM). Molecular & cellular proteomics : MCP, 2015. 14(6): p. 1630 - 1644.
[7].Martinez, E., et al., Targeted proteomics identifies proteomic signatures in liquid-biopsies of the endometrium to diagnose endometrial cancer and assist in the prediction of the optimal surgical treatment. Clinical Cancer Research An Official Journal of the American Association for Cancer Research, 2017. 23(21): p. clincanres.0474.2017.
[8].Thomas, S.N., et al., Multiplexed Targeted Mass Spectrometry-Based Assays for the Quantification of N-Linked Glycosite-Containing Peptides in Serum. Analytical Chemistry, 2015. 87(21): p. 10830-8.
[9].Xue, T., et al., Interleukin-6 Induced “Acute” Phenotypic Microenvironment Promotes Th1 Anti-Tumor Immunity in Cryo-Thermal Therapy Revealed By Shotgun and Parallel Reaction Monitoring Proteomics. Theranostics, 2016. 6(6): p. 773-794.
[10].Rauniyar, N., et al., Data-Independent Acquisition and Parallel Reaction Monitoring Mass Spectrometry Identification of Serum Biomarkers for Ovarian Cancer. Biomarker Insights, 2017. 12: p. 117727191771094.
[11].Escher, C., et al., Using iRT, a normalized retention time for more targeted measurement of peptides. PROTEOMICS, 2012. 12(8): p. 1111-1121.
[12].Piazza, I., et al., A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication. Cell, 2018. 172(1-2): p. 358-372.e23.
[13].Reitsma, J.M., et al., Composition and Regulation of the Cellular Repertoire of SCF Ubiquitin Ligases. Cell, 2017. 171(6): p. 1326-1339.e14.
[14].von Ziegler, L.M., et al., Subregion-Specific Proteomic Signature in the Hippocampus for Recognition Processes in Adult Mice. Cell Reports, 2018. 22(12): p. 3362-3374.
[15].Latonen, L., et al., Integrative proteomics in prostate cancer uncovers robustness against genomic and transcriptomic aberrations during disease progression. Nature Communications, 2018. 9(1).
[16].Smith, P.K., et al., Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 1985. 150(1): p. 76-85.
[17].Candiano, G., et al., Blue silver: A very sensitive colloidal Coomassie G‐250 staining for proteome analysis. Electrophoresis, 2010. 25(9): p. 1327-1333.
[18].2xiRT Kit Quick Reference Card.
[19].MacLean, B., et al., Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics, 2010. 26(7): p. 966-968.
[20].Cox, J. and M. Mann, MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnology, 2008. 26(12): p. 1367-1372.
[21].Escher C, Reiter L, MacLean B, Ossola R, Herzog F, Chilton J, MacCoss M.J, Rinner O: Using iRT, a normalized retention time for more targeted measurement of peptides, Proteomics 2012, 12(8): 1111-1121

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