Whitelegge Julian
The Pasarow Mass Spectrometry Laboratory, Jane and Terry Semel Institute of Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 USA.
Proteomes. 2020 Mar 10;8(1):5. doi: 10.3390/proteomes8010005.
A subsection of integral membrane proteins partition into chloroform during a chloroform/methanol/water extraction primarily designed to extract lipids. Traditionally, these proteins were called proteolipids due to their lipid-like properties; the c-subunit of the ATP synthase integral FO component is the best known due to its abundance. In this manuscript, we investigate purification of proteolipid proteins away from lipids for high-resolution mass spectrometry. Size-exclusion chromatography on silica beads using a chloroform/methanol/aqueous formic acid (4/4/1; /) mobile phase allowed the separation of larger proteins (>3 kDa) from lipids (<1.5 kDa) and analysis by online electrospray ionization mass spectrometry. Fraction collection for mass spectrometry was limited by presence of plasticizers and other contaminants solubilized by chloroform. Drying down of the protein sample followed by resuspension in formic acid (70%) allowed reverse-phase chromatography on a polymeric support at elevated temperature, as described previously. Fractions collected in this way could be stored for extended periods at -80 °C without adducts or contaminants. Top-down mass spectrometry enabled the definition of PsaI as a novel proteolipid of spinach thylakoid membrane. Proteolipid preparation worked similarly when total membranes from mouse brains were extracted with chloroform. While it might be tempting to use the described extraction, we prefer to broaden the meaning of the term, whereby the is defined as a novel biological membrane proteome that includes the full complement of membrane proteins, their binding partners/ligands and their tightly bound structural lipids that constitute each protein-lipid complex's functional unit; that is, a complete description of a biological membrane.
在主要用于提取脂质的氯仿/甲醇/水萃取过程中，一部分整合膜蛋白会分配到氯仿中。传统上，由于这些蛋白质具有类脂性质，它们被称为蛋白脂质；ATP合酶整合FO组分的c亚基因其含量丰富而最为人所知。在本手稿中，我们研究了从脂质中纯化蛋白脂质蛋白以用于高分辨率质谱分析。使用氯仿/甲醇/甲酸水溶液（4/4/1；/）流动相在硅胶珠上进行尺寸排阻色谱，可将较大的蛋白质（>3 kDa）与脂质（<1.5 kDa）分离，并通过在线电喷雾电离质谱进行分析。质谱分析的馏分收集受到增塑剂和氯仿溶解的其他污染物的限制。如前所述，将蛋白质样品干燥后再悬浮于甲酸（70%）中，可在高温下在聚合物支持物上进行反相色谱。以这种方式收集 的馏分可在-80°C下长期保存而无加合物或污染物。自上而下的质谱分析确定PsaI为菠菜类囊体膜的一种新型蛋白脂质。当用氯仿从小鼠脑提取总膜时，蛋白脂质制备方法同样有效。虽然可能很想使用所述的提取方法，但我们更倾向于拓宽该术语的含义，由此将 定义为一种新型生物膜蛋白质组，它包括膜蛋白的完整补充、它们的结合伴侣/配体以及构成每个蛋白质-脂质复合物功能单元的紧密结合的结构脂质；也就是说，对生物膜的完整描述。
