TITLE: Structural analysis of the GPI glycan
AUTHORS: Miyako Nakano †, Susana Sabido-Bozo †, Kouta Okazaki, Auxiliadora Aguilera-Romero, Sofia Rodriguez-Gallardo, Alejandro Cortes-Garcia, Sergio Lopez, Atsuko Ikeda, Kouichi Funato, Manuel Muñiz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes an optimized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis, and analysis by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm the predicted GPI-anchoring of individual proteins.</div></div>

[STEPS]
?. [Yeast growth and culture]
Pick up the yeast cells expressing the plasma membrane GPI-AP Gas1-GFP from a frozen stock using a sterile toothpick, streak them on an SD medium without leucine (SD-LEU) agar plate and incubate them at 24 ºC for 2-3 days.
?. [Yeast growth and culture]
Inoculate the yeast cells into 4 ml SD-LEU medium and growth them for 6-8 h to the early-to-mid logarithmic phase at 24 ºC with shaking at 500 rpm.
?. [Yeast growth and culture]
Dilute yeast cells into 600 - 800 ml YPD medium and growth them to mid-log phase overnight at 24 ºC with shaking at 500 rpm.
?. [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 the pellet in 12 ml of TNE precooled at 4 ºC and distribute aliquots of 1 ml into 2 ml screw-cap microcentrifuge tubes (12 tubes).
?. [Yeast growth and culture]
Centrifuge aliquots at 13000 x g for 1 min at 4 ºC, discard supernatants, and freeze the cell pellets at -80 ºC.
?. [Cellular membrane fractionation]
Quickly thaw the cell pellets and immediately place the tubes on ice.
?. [Cellular membrane fractionation]
Resuspend each cell pellet with 700 µl of prechilled TNE.
?. [Cellular membrane fractionation]
Add 600 µl of glass beads per tube.
?. [Cellular membrane fractionation]
Lyse mechanically the cells using a bead beater system. For Fastprep device: 3 cycles of 20 sec at 5 m/s. Rest on ice for 3 min between cycles.
?. [Cellular membrane fractionation]
Spin down beads and cells debris at 1000 x g for 10 min at 4 ºC and transfer supernatants into 1,5 ml UltraClear microcentrifuge tubes from Axygen (UC tube) (item 12 from the list of materials).
?. [Cellular membrane fractionation]
Spin down cellular membranes at 17000 x g for 1h at 4 ºC.
?. [Cellular membrane fractionation]
Discard the supernatant and resuspend each membrane pellet in 100 µl of TNE.
?. [Cellular membrane fractionation]
Collect the resuspended membrane pellets into two UC tubes.
?. [Cellular membrane fractionation]
Spin down the membrane pellets at 17000 x g for 1h 4 ºC.
?. [PI-PLC treatment]
Resuspend each membrane pellet in 440 µl TNE and collect them into a UC tube.
?. [PI-PLC treatment]
Add 10 µl of PI-PLC (Invitrogen 0,1U/µl) and 50 µl TNE-D 0,2%.
?. [PI-PLC treatment]
Incubate at 37 ºC for 1h.
?. [PI-PLC treatment]
Spin down the membranes at 17000 x g for 30min at 4 ºC.
?. [PI-PLC treatment]
Discard the membrane pellet and save the supernatant into a UC tube at 4 ºC.
?. [Immunoprecipitation (IP)]
Prepare 30% GFP-Trap_A beads by vortexing to remove aggregates then washing in TNE twice before resuspending in TNE to a concentration of 30%.
?. [Immunoprecipitation (IP)]
Add 100 µl of GFP-Trap beads slurry into a new UC tube (IP tube).
?. [Immunoprecipitation (IP)]
Add the 450 µl of the previously saved supernatant (step 19) to the IP tube from step 21.
?. [Immunoprecipitation (IP)]
Incubate the IP tube rotating at 4 ºC overnight.
?. [Immunoprecipitation (IP)]
The next day, centrifuge the IP tube at 5000 x g for 30 sec at 4 ºC to spin down the beads and discard the supernatant.
?. [Immunoprecipitation (IP)]
Resuspend the beads pellet with 500 µl of TNE-D 0,2% and transfer to a new UC tube.
?. [Immunoprecipitation (IP)]
Wash the original IP tube from step 24 with 500 µl of TNE-D 0,2% and combine the beads suspension with the first one into the new UC tube from step 25.
?. [Immunoprecipitation (IP)]
Centrifuge at 5000 x g for 30 s at 4 ºC and remove the supernatant.
?. [Immunoprecipitation (IP)]
Add 750 µl of TNE-D 0.2%. Repeat steps 27 and 28 three times more.
?. [Immunoprecipitation (IP)]
Centrifuge at 5000 x g for 30 s at 4 ºC and remove the supernatant. Centrifuge again to remove the remaining liquid and dry the beads using a white micropipette tip.
?. [Immunoprecipitation (IP)]
Add 40 µl of sample buffer 2x.
?. [Immunoprecipitation (IP)]
Heat the samples at 95 ºC for 5 min.
?. [Immunoprecipitation (IP)]
Vortex. Centrifuge maximum speed for 5 min at room temperature.
?. [Immunoprecipitation (IP)]
Load the supernatant onto a 7.5 % acrylamide gel and separate the immunoprecipitated Gas1-GFP by SDS-PAGE gel electrophoresis.
?. [Immunoprecipitation (IP)]
Stain the polyacrylamide gel with coomassie brilliant blue (CBB).
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Excise a gel band (~2 mm × 8 mm) of protein stained with CBB from the polyacrylamide gel.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Cut the gel band into ~1 mm3 pieces and put the gel pieces in a 1,5 ml polypropylene Safe Lock microcentrifuge tube from Eppendorf (P tube) (item 27 from the list of materials).
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 μl of the destaining solution to the P tube.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Mix the sample on a shaker continuously for 20 min at room temperature. After 20 min of mixing, remove and discard the liquid using a pipette.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Repeat steps 37 and 38 one or two more times until removing CBB from gel pieces.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of acetonitrile (CH3CN) to the gel pieces.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Mix the sample on a shaker continuously for 10 min at room temperature. After 10 min of mixing, remove and discard the liquid. The gel pieces will shrink and become opaque with this treatment.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of the fresh reduction solution to the shrinking and opaque gel pieces in the sample P tube and incubate the P tube at 65 ºC for 1 hour. After 1 h incubation, remove and discard the liquid.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of the fresh alkylation solution to the gel pieces in the sample P tube and incubate the P tube in the dark at room temperature for 30 min. After 30 min of incubation, remove and discard the liquid.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of wash solution (50 mM NH4HCO3) to the sample P tube.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Mix the tube on a shaker continuously for 10 min at room temperature. After 10 min of mixing, remove and discard the liquid.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Repeat steps 44 and 45 one more time.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of acetonitrile (CH3CN) to the gel pieces.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Mix the sample on a shaker continuously for 10 min at room temperature. After 10 min of mixing, remove and discard the liquid. The gel pieces will shrink and become opaque with this treatment.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add trypsin solution to the sample P tube at 1/20–1/100 enzyme(ng): substrate(ng) with the minimum volume that the swelled gel can cover.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Preincubate the sample for 5 min at room temperature.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add digestion solution (50 mM NH4HCO3) to the sample P tube to completely cover the gel pieces.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Incubate the sample P tube at 37 ºC overnight.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Suck and transfer the liquid to a new P tube using a pipette.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of extraction solution 1 to the gel pieces.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Sonicate the sample P tube containing gel pieces for 10 min at room temperature using float.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Suck and transfer the liquid to the P tube from step 53 using a pipette.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Add 200 µL of extraction solution 2 to the gel pieces.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Sonicate the P tube containing gel pieces for 10 min at room temperature using float.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Suck and transfer the liquid to the P tube from step 53 using a pipette.
?. [Preparation of the sample for mass spectrometry (MS) analysis]
Dry the combined liquid in the P tube from 53 with a Speed Vac at room temperature for 2–4 hours (do not use heating). The dried samples can be stored in the freezer prior to LC-ESI MS analysis.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Dissolve the dried residue with 20 µL of ultrapure water for LC-ESI MS analysis.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Centrifuge at 13,200 x g for 5 min at 4°C and transfer 12 µL of the supernatant to a new vial for Accela autosampler.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Inject 8 µL of sample solution into LC.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Separate the peptides bearing GPI-glycans using an ODS column under specific gradient conditions.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Elute the column with solvent A for 5 min, at which point increases the concentration of solvent B to 40 % over 55 min at a flow rate of 50 µL/min using LC system.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Introduce continuously the eluate into an ESI source.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Analyze the Peptides bearing GPI-glycans by LTQ Orbitrap XL.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Set in the MS setting the voltage of the capillary source at 4.5 kV,  and maintain the temperature of the transfer capillary at 300 ºC.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Set the capillary voltage and tube lens voltage at 15 and 50 V, respectively.
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Obtain MS data in positive ion mode over the mass range m/z 300 to m/z 3000 (resolution: 60000, mass accuracy: 5 ppm).
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Obtain MS/MS data by ion trap in LTQ Orbitrap XL (data dependent top 3, CID).
?. [Mass spectrometry analysis of the peptides bearing GPI glycans]
Analyze the MS and MS/MS data to identify the structure of GPI-glycans (See Fig. 2 and 3 of the associated article).