Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors
Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors
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DOI：
10.1189/jlb.1212619
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发表时间：
2013-10-01
影响因子：
5.5
通讯作者：
Borregaard, Niels
中科院分区：
文献类型：
--
作者：
Rorvig, Sara;Ostergaard, Ole;Borregaard, Niels
中性粒细胞在抵抗入侵微生物的先天免疫防御中是不可或缺的。中性粒细胞含有SV和几个对其功能至关重要的颗粒子集。存在于中性粒细胞SV和颗粒中的蛋白质在骨髓中的终末粒细胞生成期间合成。颗粒的异质性，由每个颗粒子集的标志物蛋白质特征确定，被认为是由于主要类别的颗粒蛋白质的生物合成窗口的差异，一个被称为定时靶向的过程。中性粒细胞颗粒、SV和质膜的定性蛋白质组学分析以前已经进行过。在这里，我们进行了亚细胞分馏新鲜分离的人中性粒细胞的氮空化和密度离心上的四层Percoll梯度。将颗粒亚组合并并进行SDS-PAGE，并将凝胶片用胰蛋白酶进行凝胶内消化。使用LTQ Orbitrap XL串联MS分析所得肽。根据中性粒细胞分数，共鉴定并分组了1292种独特的蛋白质，其中它们显示最大表达。除了各种已知的中性粒细胞蛋白质，发现了几个未知的蛋白质，以及蛋白质没有以前描述的中性粒细胞。为了研究中性粒细胞前体mRNA表达与其同源蛋白定位之间的相关性，将126种鉴定的蛋白的分布与其mRNA表达谱进行比较。本文提供的中性粒细胞亚细胞蛋白质组谱可用作与mRNA阵列数据库组合的数据库，以预测和测试中性粒细胞颗粒和膜中蛋白质的存在和定位。
Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting pep-tides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes.