TITLE: Determination of the lipid composition of the GPI anchor
AUTHORS: Auxiliadora Aguilera-Romero †, Susana Sabido-Bozo †, Sergio Lopez, Alejandro Cortes-Garcia, Sofia Rodriguez-Gallardo, Ana Maria Perez-Linero, Isabelle Riezman, Howard Riezman, Manuel Muñiz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In eukaryotic cells, a subset of cell surface proteins is attached by the glycolipid glycosylphosphatidylinositol (GPI) to the external leaflet of the plasma membrane where they play important roles as enzymes, receptors, or adhesion molecules. Here we present an improved protocol for purification and mass spectrometry analysis of the lipid moiety of individual GPI-anchored proteins (GPI-APs) in yeast. The method involves the expression of a specific GPI-AP tagged with GFP, solubilization, immunoprecipitation, separation by electrophoresis, blotting onto PVDF, release, extraction of the GPI-lipid moiety, and analysis by mass spectrometry. By using this protocol, we could determine the precise GPI-lipid structure of the GPI-AP Gas1-GFP in a modified yeast strain. This protocol can be used to identify the lipid composition of the GPI-anchor of distinct GPI-APs from yeast to mammals and can be adapted to determine other types of protein lipidation. </div></div>

[STEPS]
?. [Yeast growth and culture]
Transform the yeast strain with a centromeric plasmid expressing GFP-tagged Gas1p under control of its own promoter (pRS416-GAS1-GFP).
?. [Yeast growth and culture]
Pick up a colony and streak it on a SD agar plate with appropriate nutritional supplements (SC-URA) and incubate them at 24 °C for 2-3 days.
?. [Yeast growth and culture]
Inoculate transformed yeast cells in 3ml of SC-URA in a 12 ml sterile tube and growth them to the early-to-mid logarithmic phase at 24 °C with shaking at 250 rpm.
?. [Lipid extraction from GPI anchor]
Wash PVDF membrane strips slowly avoiding touching the membrane with 1ml of methanol (MS quality) four times.
?. [Lipid extraction from GPI anchor]
Wash PVDF strips with 1ml of water (MS quality).
?. [Lipid extraction from GPI anchor]
Incubate with a filtered mixture of 500 μl of 0.3 M NaOAc pH 4.0 buffer and 500 μl of freshly dissolved 1 M NaNO2 for 3 h at 37 °C.
?. [Lipid extraction from GPI anchor]
Remove the solution and wash the PVDF membrane strips with 1 ml of water MS-LC grade four times.
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Extract the GPI-lipids from the PVDF strip with 75 μl of negative mode solvent (Chloroform/Methanol (1:2) + 5 mM Ammonium acetate).
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Transfer the sample into a 96 well plate and seal with an Easy Pierce 20 µm Foil to avoid evaporation of the sample.
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Infuse the sample on a TSQ Vantage using a Triversa Nanomate equipped with a HD_A chip with a gas pressure of 30 psi and a spray voltage of 1.2 kV.
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Operate the TSQ Vantage with a spray voltage of 3 kV in negative-ion mode. The capillary temperature is set to 190 °C.
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Use Multiple Reaction Monitoring (MRM) to obtain the signal intensity for each lipid molecular species (Fragmentation is performed with a collision energy optimized for each lipid class)
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Read 3 times the MRM list (Table 1) on the TSQ Vantage to obtain three technical replicates for each biological replicate of the strain (It is advisable, to include at least, 3 biological replicates per strain) The MRM measurements are done with a width set to 1.0 in the Q3.
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Calculate the signal intensity for each parent ion adding the intensities obtained in a range of ± 0.5 the m/z of the production. This can be done manually with Thermo Xcalibur software.
?. [Mass spectrometric analysis of IPC from Gas1-GFP]
Finally, calculate the proportional signal for each lipid species compared to the sum of all the signals.
?. [Yeast growth and culture]
Dilute yeast cells into 800 ml of YPD medium and growth them to mid-log phase overnight at 30°C with shaking at 250 rpm. YPD medium is required for the correct expression of Gas1-GFP at the cell surface.
?. [Yeast growth and culture]
On the next day, harvest 600 x 107 cells by centrifugation at 3000 x g for 5 min, discard the supernatant, resuspend in 12 ml of TNE precooled at 4°C and distribute aliquots of 1 ml into 2 ml screw-cap tubes (12 tubes).
?. [Yeast growth and culture]
Centrifuge aliquots at 13,000 x g for 1 min, discard supernatant and freeze the pellet at -80 °C.
?. [Immunoprecipitation of Gas1-GFP]
Break cells using Fast Prep (3 pulses of 30 s at 5 m/s).
?. [Immunoprecipitation of Gas1-GFP]
Remove glass beads and cell debris by centrifugation at 1000 x g for 10 min at 4 °C.
?. [Immunoprecipitation of Gas1-GFP]
Transfer 1 ml of supernatant into a 1.5 ml ultraclear tube (UC).
?. [Immunoprecipitation of Gas1-GFP]
Centrifuge at 17,000 x g for 20 min at 4 °C.
?. [Immunoprecipitation of Gas1-GFP]
Resuspend and combine all the pellets into a single 1.5 ml UC tube using 1 ml of TNE.
?. [Immunoprecipitation of Gas1-GFP]
Add 250µl of TNE-D 5 % to obtain a solution with a final concentration of 1% digitonin. Mix with end-over-end rotation for 1 h at 4 °C.
?. [Immunoprecipitation of Gas1-GFP]
Centrifuge the suspension at 17,000 x g for 20 min at 4 °C to remove insoluble material.
?. [Immunoprecipitation of Gas1-GFP]
Transfer the supernatant to an UC tube with 100 µl GFP-Trap beads at  (30 % slurry) and incubate overnight at 4 °C.
?. [Immunoprecipitation of Gas1-GFP]
Next day, centrifuge at 5,000 x g for 30 s at 4 °C and remove supernatant.
?. [Immunoprecipitation of Gas1-GFP]
Resuspend the beads pellet with 500 µl of TNE-D 1 % and transfer to a new UC tube.
?. [Immunoprecipitation of Gas1-GFP]
Wash the original UC tube with 500 µl of TNE-D 1 % and combine the beads suspension with the first one.
?. [Immunoprecipitation of Gas1-GFP]
Centrifuge at 5,000 x g for 30 s at 4 °C and remove the supernatant.
?. [Immunoprecipitation of Gas1-GFP]
Add 750 µl TNE-D 0.2 %. Repeat step 19 and 20 twice more.
?. [Immunoprecipitation of Gas1-GFP]
Centrifuge at 5,000 x g for 30 s at 4 °C and remove the supernatant. Centrifuge again to remove the remaining liquid and dry the beads using a white micropipette tip.
?. [Immunoprecipitation of Gas1-GFP]
Add 40 µl of SB 2x.
?. [Immunoprecipitation of Gas1-GFP]
Heat the samples at 95 °C for 10 min.
?. [Immunoprecipitation of Gas1-GFP]
Vortex. Centrifuge maximum speed for 5 min at room temperature.
?. [Immunoprecipitation of Gas1-GFP]
Load the supernatant onto a 7.5 % acrylamide gel, separate the proteins by SDS-PAGE gel electrophoresis and transfer to a PVDF membrane previously activated by methanol (Wet the PVDF membrane in 100 % methanol for a few seconds and then equilibrate the membrane in transfer buffer for a few minutes until it sinks).
?. [Immunoprecipitation of Gas1-GFP]
After transfer, stain the PVDF membrane with Amido Black Staining Solution 2x for 1min.
?. [Immunoprecipitation of Gas1-GFP]
Wash five times with the destaining solution for 2 min.
?. [Immunoprecipitation of Gas1-GFP]
Cut out Gas1-GFP band with a new razor blade and transfer to an amber glass tube or screw-cap Eppendorf tube.
?. [Immunoprecipitation of Gas1-GFP]
Add 300 µl of glass beads and 1.5 ml of TNE to each cell pellet.
?. [Lipid extraction from GPI anchor]
Dry and transfer the membrane to an amber tube. Flush the samples with N2 and store at -80 °C.