OFFgel-based multidimensional LC-MS/MS approach to the cataloguing of the human platelet proteome for an interactomic profile
OFFgel-based multidimensional LC-MS/MS approach to the cataloguing of the human platelet proteome for an interactomic profile
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DOI：
10.1002/elps.201000592
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发表时间：
2011-03-01
期刊：
影响因子：
2.9
通讯作者：
Cuda, Giovanni
中科院分区：
文献类型：
--
作者：
Krishnan, Shibu;Gaspari, Marco;Cuda, Giovanni
通过鸟枪法蛋白质组学方法分析静止人血小板的蛋白质组，该方法包括酶消化、基于等电点通过使用OFFgel分级分离的肽分离以及最后的RP纳米级色谱与MS/MS检测耦合（nano-LC-MS/MS）。在第一维中的OFFgel分级在提供与RP nano-LC正交的额外分离维度方面是有效的，从而产生经证明是稳健且易于建立的离线多维分离平台。分析鉴定了1373个具有高置信度的蛋白质（错误发现率< 0.25%）。通过Inflamity Pathway Analysis软件研究了1373种人类血小板蛋白的核心集，其中10种经典途径和8种网络已经得到验证，表明血小板表现为炎症或免疫细胞，质膜和细胞骨架蛋白在其功能中起着重要作用。此外，与网络分析一致的毒性途径支持血小板寿命受凋亡机制支配的概念。
The proteome of quiescent human platelets was analyzed by a shotgun proteomics approach consisting of enzymatic digestion, peptide separation based on isoelectric point by the use of OFFgel fractionation and, finally, RP nanoscale chromatography coupled to MS/MS detection (nano-LC-MS/MS). OFFgel fractionation in the first dimension was effective in providing an additional dimension of separation, orthogonal to RP nano-LC, thus generating an off-line multidimensional separation platform that proved to be robust and easy to set up. The analysis identified 1373 proteins with high confidence (false discovery rate < 0.25%). The core set of 1373 human platelet proteins was investigated by Ingenuity Pathway Analysis software from which ten canonical pathways and eight networks have been validated, to suggest that platelets behave either as inflammatory or immune cells, and plasma membrane and cytoskeleton proteins play a fundamental role in their function. Moreover, toxicity pathway in agreement with network analysis, supports the concept that platelet life span is governed by an apoptotic mechanism.