1 Isolation of Membrane Proteins Using Triton X-114
1.1 Triton X-114 Phase Partitioning
(a) To obtain the cell lysate, approximately 4 million cells are homogenized in Triton lysis buffer containing a protease inhibitor cocktail to prevent protein degradation. Ultrasonic processor is used for homogenization and samples are then incubated at 4°C for an hour. To get a clear lysate, it is then subjected to centrifugation for 30 minutes at 10,000 x g at 4°C.
(b) The resulting clear supernatant is then overlaid onto a sucrose cushion and incubated at 37°C for 20 minutes, resulting in a cloudy solution. Samples are then centrifuged for 3 minutes at 400 x g and 37°C, resulting in the formation of two phases, a detergent-rich and aqueous detergent-poor phase.
(c) Transfer the aqueous phase to a fresh tube and keep it on ice.
(d) Resuspend the detergent phase in 500 μL of cold PBS and repeat the phase separation once again. Pool this aqueous phase with initial one and re-extract by adding 50 μL of Triton stock solution to perform the phase separation.
1.2 Protein Precipitation
Since presence of Triton X-114 interferes with enzymatic deglycosylation with N -glycosidase F, an amidase, also known as PNGase F, which cleaves between innermost GlcNAc of N - glycan and asparagine residue on protein, it must be removed prior to glycan analysis . The best method is precipitation with organic solvents. The method of choice, which is described below, is the chloroform– methanol–water precipitation.
(a) Add four times the sample volume of methanol to each sample and vortex well.
(b) Sequentially, add twice the initial sample volume of chloroform to each sample and vortex well.
(c) At the end, add three times of the initial sample volume of water, vortex vigorously and centrifuge at 9000 × g for 1 min at 4 °C. After centrifugation the proteins are located in the liquid interphase.
(d) Remove the aqueous top layer, add additional three volumes of methanol, vortex samples and centrifuge again for 2 min at 9000 × g, 4 °C to pellet the proteins.
(e) Remove the supernatant as much as possible, without disturbing the precipitate, and leave samples to air-dry.
2 Analysis of N-linked Glycans Using HILIC-UPLC or HPLC
2.1 Glycan Release and Labeling
(a) Resuspend pellets by adding 30 μL of 1.33 % SDS (w/v) and incubate at 65 °C for 10 min.
(b) Subsequently, add 10 μL of 4 % Igepal-CA630 and 1.25 mU of PNGase F in 10 μL of 5× PBS to each sample. Incubate the samples overnight at 37 °C for N -glycan release.
(c) Now prepare labeling mixture as indicated in Subheading b. Always prepare this mixture FRESH.
(d) Add 25 μL of labeling mixture to each sample, followed by 2 h incubation at 65 °C.
(e) Add 700 μL of ACN to each sample in order to bring it to 96 % ACN (v/v).
(f) Prepare the 0.2 μm GHP 96-well filter plate. For this you need to prewash the plate with 200 μL of 70 % ethanol (v/v) and then 200 μL of water, followed by equilibration with 200 μL of 96 % ACN (v/v).
(g) Transfer the samples to the prepared plate by using multichannel pipette. Remove the solvent using vacuum.
(h) Wash loaded samples 5× with 200 μL of 96 % ACN (v/v) following the same procedure for loading and removing the solution as described in Subheading 2 (preparation of the filter plate on vacuum manifold). Elute glycans with 2 × 90 μL water and store at −20 °C until chromatographic analysis.
2.2 Hydrophilic Interaction Liquid Chromatography (HILIC)-UPLC/HPLC
After labeling with a fluorescent tag, the N-glycans can be separated by hydrophilic interaction chromatography using either a UPLC or HPLC instrument with a fluorescence detector set at excitation and emission wavelengths of 250 and 420 nm, respectively. For UPLC, separation is performed on a BEH Glycan chromatography column with a linear gradient of 75-53% acetonitrile and 100 mM ammonium formate, pH 4.4, as solvent A and B, respectively. The run time is 25 minutes, and the samples are kept at 5°C before injection. For HPLC, separation is performed on a TSKgel Amide 80 column with a linear gradient of 65-53% acetonitrile and 50 mM ammonium formate, pH 4.4, as solvent A and B, respectively. The run time is 60 minutes, and the column is maintained at 30°C. In both cases, the chromatographic areas are integrated to determine the amount of glycans in each peak, which is expressed as a percentage of the total integrated area.
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Reference
- Lauc, G., & Wuhrer, M. (2017). High-throughput glycomics and glycoproteomics. Springer New York.