SILAC-iPAC: a quantitative method for distinguishing genuine from non-specific components of protein complexes by parallel affinity capture.
SILAC-iPAC: a quantitative method for distinguishing genuine from non-specific components of protein complexes by parallel affinity capture.
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DOI：
10.1016/j.jprot.2014.12.006
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发表时间：
2015-02-06
影响因子：
3.3
通讯作者：
Jackson AP
中科院分区：
文献类型：
--
作者：
Rees JS;Lilley KS;Jackson AP
下拉测定法可以鉴定蛋白质复合物的成员，但遭受污染物的共分离。当特异性相互作用伴侣具有低丰度和/或以低亲和力瞬时结合时，该问题特别严重。为了区分真正的相互作用的合作伙伴从污染物，我们结合了SILAC标记与蛋白质组学方法称为“相互作用组平行亲和捕获”（iPAC）。在我们的方法中，稳定表达双标记的靶内源性蛋白的细胞系及其无标记的对照细胞系被差异SILAC标记。将来自两种细胞系的裂解物混合，并使用多个亲和树脂平行地独立纯化标记的蛋白质用于MS分析。这允许定量鉴定标记的蛋白质及其结合配偶体。SILAC-iPAC提供了一种严格而灵敏的方法，即使下拉中的污染物大量过量，也可以区分真正的结合伴侣和污染物。我们用我们的方法检测了鸡前B细胞系DT 40中磷脂酰肌醇5-磷酸4-激酶2β亚基（PI 5 P4 K2 β）和范可尼贫血核心复合物的相互作用伴侣。我们确认了这两种复合物的已知成分，并确定了新的潜在结合伙伴。将iPAC方法与SILAC标记相结合提供了一种灵敏且完全定量的方法，用于在低信噪比不可避免的条件下区分特定相互作用。此外，我们的工作提供了在DT 40蛋白质组和非特异性DT 40“珠粒”（结合到珠粒的非特异性蛋白质）的共同表位标签内的最丰富的蛋白质的第一表征。鉴于DT 40细胞系的重要性和广泛使用，这些细胞将成为细胞生物学和免疫学社区的重要资源。生物学意义SILAC-iPAC为分析低亲和力和/或低丰度蛋白质-蛋白质相互作用提供了一种改进的方法。我们用它来澄清两个例子，其中蛋白质复合物的性质是已知的，或目前尚不清楚。该方法简便、定量，适用于细胞和分子生物学中的许多问题。我们还报道了第一批鸡珠。SILAC-iPAC;定量检测低丰度蛋白质RUVBL 1及其配偶体RUVBL 2的改进的AP-MS方法是范可尼贫血复合体的新的相互作用物。使用四个共同表位标签首次表征鸡DT 40珠粒
Pull-down assays can identify members of protein complexes but suffer from co-isolation of contaminants. The problem is particularly acute when the specifically interacting partners are of low-abundance and/or bind transiently with low affinity. To differentiate true interacting partners from contaminants, we have combined SILAC labelling with a proteomic method called “Interactomes by Parallel Affinity Capture” (iPAC). In our method, a cell-line stably expressing a doubly tagged target endogenous protein and its tag-less control cell-line are differentially SILAC labelled. Lysates from the two cell-lines are mixed and the tagged protein is independently purified for MS analysis using multiple affinity resins in parallel. This allows the quantitative identification of tagged proteins and their binding partners. SILAC–iPAC provides a rigorous and sensitive approach that can discriminate between genuine binding partners and contaminants, even when the contaminants in the pull-down are in large excess. We employed our method to examine the interacting partners of phosphatidyl inositol 5-phosphate 4-kinase 2β subunit (PI5P4K2β) and the Fanconi anaemia core complex in the chicken pre-B cell-line DT40. We confirmed known components of these two complexes, and we have identified new potential binding partners. Combining the iPAC approach with SILAC labelling provides a sensitive and fully quantitative method for the discrimination of specific interactions under conditions where low signal to noise ratios are unavoidable. In addition, our work provides the first characterisation of the most abundant proteins within the DT40 proteome and the non-specific DT40 ‘beadomes’ (non-specific proteins binding to beads) for common epitope tags. Given the importance and widespread use of the DT40 cell-line, these will be important resources for the cell biology and immunology communities. Biological significance SILAC–iPAC provides an improved method for the analysis of low-affinity and/or low abundance protein-protein interactions. We use it to clarify two examples where the nature of the protein complexes are known, or are currently unclear. The method is simple and quantitative and will be applicable to many problems in cell and molecular biology. We also report the first chicken beadomes. SILAC–iPAC; an improved AP-MS method to quantitatively detect low abundance proteins RUVBL1 and its partner RUVBL2 are novel interactors of the Fanconi anaemia complex. First characterisation of chicken DT40 beadomes using four common epitope tags
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影响因子：
3.3
作者：
Altelaar, A. F. Maarten;Frese, Christian K.;Mohammed, Shabaz
通讯作者：
Mohammed, Shabaz
DOI：
10.1074/mcp.m110.002386
发表时间：
2011-06
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Rees JS;Lowe N;Armean IM;Roote J;Johnson G;Drummond E;Spriggs H;Ryder E;Russell S;St Johnston D;Lilley KS
通讯作者：
Lilley KS
影响因子：
14.9
作者：
Franceschini A;Szklarczyk D;Frankild S;Kuhn M;Simonovic M;Roth A;Lin J;Minguez P;Bork P;von Mering C;Jensen LJ
通讯作者：
Jensen LJ
影响因子：
3
作者：
Dazard JE;Saha S;Ewing RM
通讯作者：
Ewing RM
影响因子：
4.8
作者：
Richardson, Jonathan P.;Wang, Minchuan;Irvine, R. F.
通讯作者：
Irvine, R. F.