Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan.
Fluids Barriers CNS. 2013 Jun 8;10(1):21. doi: 10.1186/2045-8118-10-21.
Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain "black boxes". Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography-tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx).
蛋白质组学为生物科学开辟了新的视野。 全面蛋白质组分析是一种很有前途的技术，可用于发现各种生物系统中的数千种蛋白质、翻译后修饰、多态性和分子相互作用。 还必须阐明鉴定出的蛋白质的活性和作用，但由于常规蛋白质组学方法无法提供蛋白质表达的定量信息，因此这变得很复杂。 因此，许多生物系统仍然是“黑盒子”。 定量靶向绝对蛋白质组学 (QTAP) 可用于确定任何靶蛋白的绝对表达水平 (mol)，包括低丰度功能蛋白，如转运蛋白和受体。 因此，QTAP 将有助于理解单个蛋白质及其差异的活性和作用，包括正常/疾病、人和动物或体外/体内。 在这里，我们描述了 QTAP 实验的研究方案和注意事项，包括靶肽的计算机选择、氨基酸分析测定肽浓度、在液相色谱-串联质谱中建立选定的/多重反应监测 (SRM/MRM) 分析、蛋白样品的制备（脑毛细血管和质膜部分），然后是肽样品的制备，通过 SRM/MRM 分析同时对靶蛋白进行绝对定量，数据分析和故障排除。 介绍了 QTAP 在生物科学中的应用，该应用利用了在 ddY、FVB 和 C57BL/6J 小鼠的转运蛋白、受体、紧密连接蛋白和血脑屏障标记蛋白的蛋白质表达水平的种间差异数据。 在 18 种分子中，13 种（abcb1a/mdr1a/P-gp、abcc4/mrp4、abcg2/bcrp、slc2a1/glut1、slc7a5/lat1、slc16a1/mct1、slc22a8/oat3、insr、lrpl、tfr1、claudin-5、Na+/K+-ATPase 和 γ-gtp）在分离的脑毛细血管中被检测到，其蛋白表达水平在 0.637-101 fmol/μg 蛋白范围内。 在这三个品系之间，水平的最大差异为 13 种分子的 2.2 倍，尽管 bcrp 和 mct1 在 C57BL/6J 和其他品系之间存在统计学上的显著差异。 QTAP 实现的高灵敏度同时绝对定量将增加蛋白质组学在生物科学中的实用性，并有望推进药物蛋白质组学 (PPx) 的新研究领域。
