Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence
Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence
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DOI：
10.1074/mcp.m112.020230
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发表时间：
2013-05-01
影响因子：
7
通讯作者：
Walther, Tobias C.
中科院分区：
文献类型：
--
作者：
Krahmer, Natalie;Hilger, Maximiliane;Walther, Tobias C.
脂滴是能量代谢和脂质储存的重要细胞器。它们的核心由形成疏水相的中性脂质组成，并被含有特定蛋白质的磷脂单层包围。大多数公认的LD蛋白执行重要的功能，特别是在细胞脂质代谢中。形态学研究表明LD与膜结合的细胞器密切相关并相互作用，包括内质网，线粒体，过氧化物酶体和内体。由于这些紧密的关联，很难将LD纯化为同质的。因此，通过蛋白质组学可靠地鉴定真正的LD蛋白一直具有挑战性。在这里，我们报告了一种基于质谱和蛋白质相关谱的LD蛋白质鉴定方法。使用LD纯化和定量，高分辨率质谱，我们确定LD蛋白相关的纯化配置文件的已知LD蛋白。从果蝇S2细胞中分离的LD的蛋白质相关性配置文件策略的应用导致111 LD蛋白质的细胞LD部分中，其中1481蛋白质被检测到的鉴定。通过表达蛋白的显微镜检查在鉴定的蛋白质的子集中确认LD定位，从而验证该方法。在所确定的LD蛋白中，既有特征明确的LD蛋白，也有以前不知道定位于LD的蛋白。我们的方法提供了一个高置信度的果蝇细胞LD蛋白质组和一种新的方法，可应用于识别LD蛋白的其他细胞类型和组织。
Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues.