Centre of Functional Genomics of Microbes, Institute of Microbiology, Department of Microbial Proteomics , University of Greifswald , Felix-Hausdorff-Strasse 8 , 17489 Greifswald , Germany.
Research & Technology , Novozymes A/S , Krogshoejvej 36 , Bagsværd DK-2880 , Denmark.
Anal Chem. 2019 Sep 17;91(18):11972-11980. doi: 10.1021/acs.analchem.9b02869. Epub 2019 Sep 3.
The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on secretion capabilities.
在过去的十年中，系统生物学领域发展迅速。然而，“组学”方法所产生的数据落后于该领域的要求，特别是在膜蛋白的绝对丰度方面。在本研究中，建立了一种新的方法，用于对革兰氏阳性模式细菌中的这种具有挑战性的蛋白质亚类进行大规模绝对定量，并使用渗透压应激管理作为原理验证先例进行了评估。选择的膜蛋白使用 SNAP 标签进行标记，这使我们能够通过免疫测定法直观地检查膜部分的富集情况。通过在染色体 SNAP 标记和野生型 菌株的粗膜提取物中加入具有已知浓度的蛋白质标准品，通过鸟枪法蛋白质组学来确定绝对膜蛋白浓度。随后，使用 SNAP 作为锚定蛋白通过靶向质谱法对鸟枪数据进行校准，并计算富集因子，以获得每平方微米的膜蛋白拷贝数。所提出的方法能够准确地确定由施加的高渗应激引起的生理变化，从而清晰地可视化膜蛋白排列的变化，并阐明假定的膜复合物。这种用于定量蛋白质组学研究的简单且具有成本效益的方法可以由任何具有质谱专业知识的研究小组实施。重要的是，它可以应用于从环境应激到小分子和蛋白质的生物技术生产等全范围的生理相关条件，这一领域严重依赖于分泌能力。
