Buck Institute for Age Research, Novato, CA 94945, USA.
Proteome Sci. 2007 Aug 10;5:12. doi: 10.1186/1477-5956-5-12.
Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods.
To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis.
Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.
多形核粒细胞（PMN）是宿主固有防御机制抵御微生物入侵的重要细胞成分，对颗粒和可溶性刺激物均表现出广泛的反应。作为急性炎症中最早募集的细胞，PMN 能够快速响应，并在接触微生物或其产物后的数分钟内释放多种有效的细胞毒性物质。PMN 依赖于功能重要的蛋白质从细胞内区室到质膜和吞噬体的重新分布，作为快速响应的手段。为了确定 PMN 激活过程中可用于快速募集的膜蛋白范围，我们分析了从小鼠静止 PMN 的光膜部分经 Percoll 梯度离心和自由流电泳纯化后获得的富含质膜和分泌小泡的亚细胞级分中的蛋白质，采用基于质谱的蛋白质组学方法。
为了鉴定富含质膜小泡和分泌小泡的光膜部分中的蛋白质，我们首先采用 MALDI-TOF（肽质量指纹图谱），然后采用 HPLC-MS/MS 分析了来自静止 PMN 的两种小泡群体，使用 3D 离子阱质谱仪。我们鉴定了几种以前未在 PMN 分泌小泡中回收的功能重要的蛋白质。其中两种蛋白质，5-脂氧合酶激活蛋白（FLAP）和 dysferlin 通过免疫印迹分析进一步验证。
我们的数据表明，在分泌小泡中存在广泛的蛋白质，为 PMN 提供了在暴露于促炎剂或刺激物后对其质膜进行显著和快速重组的能力。
