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; v
) 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 proteolipidome
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.
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