Mammalian CD1a proteins form HEK293 cells were extracted with chloroform, methanol, and water according to a modified Bligh & Dyer method as described10 (link). The lipid containing organic phase was separated from the aqueous phase and collected for the further analysis. HPLC-MS and lipidomics analysis of CD1a eluted lipids were performed as previously described10 (link), 18 (link). Briefly, extracted lipids were analyzed with an Agilent Technologies 6520 Accurate-Mass Q-TOF coupled with an Agilent 1200 series HPLC system controlled by MassHunter software. The concentration of the extracted lipids was normalized to the input proteins. Using insect-derived BK6 TCR-CD1aendo complex crystals were separated from mother liquor from approximately 50 crystallisation drops and washed with 6× 400μL crystallisation well solution using a 0.2 μm centrifugal filter at 500g. The harvested crystals were dissolved in 10 mM tris pH 8, 150 mM sodium chloride prior to mass spectrometry as described above.
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Lipidomic Analysis of CD1a Proteins
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Cd1a
Chloroform
Crystallisation
Hek293 cells
Hplc
Insect
Lipids
Liquor
Mammalian
Mass spectrometry
Methanol
Mother
Proteins
Sodium chloride
Tris
Corresponding Organization :
Other organizations : Monash University, University of Melbourne, Peter Doherty Institute, Harvard University, Brigham and Women's Hospital, ARC Centre of Excellence in Advanced Molecular Imaging, Columbia University
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Variable analysis
independent variables
- Extraction of mammalian CD1a proteins from HEK293 cells using chloroform, methanol, and water (modified Bligh & Dyer method)
- Separation of lipid-containing organic phase from aqueous phase
- HPLC-MS and lipidomics analysis of CD1a eluted lipids
- Mass spectrometry analysis of dissolved BK6 TCR-CD1aendo complex crystals
- Normalization of extracted lipid concentration to input proteins
- Washing of BK6 TCR-CD1aendo complex crystals with crystallisation well solution
- Dissolution of BK6 TCR-CD1aendo complex crystals in 10 mM Tris pH 8, 150 mM sodium chloride buffer prior to mass spectrometry
- Not explicitly mentioned
- Not explicitly mentioned
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