代謝標記技術(shù)
用疊氮基或炔基糖代謝標記細胞裂解物的制備Protocol
Metabolic Labeling of Glycoproteins 
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General Information This protocol details the preparation of cell lysates metabolically labeled with azido- or alkynylsugar. However, investigators may wish to determine the optimal concentration of metabolic reagent as well as labeling time individually for metabolic labeling for each cell type on a smallscale first.
Metabolic Labeling of Glycoproteins
Metabolic labeling is critical for successful glycoproteomics and should be carefully assessed for each cell line of interest prior to MS analysis. Metabolic labeling of glycoproteins is dependent on three considerations: first, the sugar precursor selected for labeling; second, access of the sugar precursor to the cell line; and third, glycosylation pathways activated in the cell line. Options for sugar precursor selection include Ac4ManNAz, Ac4GalNAz, Ac4GlcNAz, and Ac4ManNAl, among others. Ac4ManNAz and Ac4ManNAl are reporters of sialic acid. Ac4GalNAz and Ac4GlcNAz are able to access an intracellular epimerase pathway and are incorporated into O-GalNAc, O- GlcNAc, and N-GlcNAc sites. The efficiency of metabolic labeling is dependent on access of the sugar precursor to the cell line, and testing with several sugar precursors may be necessary for best results. Finally, sugars with access to multiple metabolic endpoints (e.g., Ac4GalNAz) will be incorporated to a set of glycoproteins that are reflective of the glycosylation pathways currently activated in the cell line. In general, human cell lines are permissive to the sugar precursor and cell counts of 107–108 (equivalent to 1–3 mg of total protein) are sufficient inputs for this protocol. In the event that metabolic labeling is prohibitive, alternate options to install bioorthogonal handles to the sample of interest may be considered (e.g., enzymatic labeling).
1.1 Prepare a stock solution of metabolic labeling reagent with DMSO to make a 500X or 1,000X stock solution. This process ensures that the DMSO concentration is not more than 0.1–0.2% in the cell culture.
1.2 Wash cells once with warm PBS or HEPES, add methionine-free medium to the cells and incubate the cells at 37°C for 30–60 minutes to deplete methionine reserves.
1.3 Add the desired concentration of metabolic labeling reagent to the cell medium, gently mix, and incubate at 37°C, 5% CO2 for 1-4 hours. Working concentrations vary from 10 μM to 100 μM. Working concentrations below or above this range are also possible, and should be optimized for metabolic labeling for each cell type.
1.4 The cells are ready for imaging, flow analysis, or harvesting.
Cell Preparation for Imaging or Flow Cytometry.
2.1 Wash the cells with PBS or HEPES to remove excess of metabolic labeling reagent.
2.2 Fix the cells with 4% paraformaldehyde in PBS for 15 minutes.
2.3 Permeabilize the cells using a fixation and permeabilization protocol of your choice. Click reaction is compatible with all protocols.
Example: 0.25% Triton? X-100 in PBS, 15 minutes or 1% BSA/0.1% saponin in PBS, 15 minutes.
2.4 Wash the cells with 3% BSA in PBS.
2.5 The cells are now ready for the click reaction with the corresponding azide- or alkyne- tagged reagent using Click-&-Go? Cell Reaction Buffer Kit.
Harvesting Suspension Cells
3.1 Pellet the cells by centrifugation at 400 × g for 5 minutes. Discard the supernatant.
3.2 Resuspend the cell pellet in PBS, gently pipette the cells up and down using 5 mL PBS for cells from a 100 mm dish or 1 mL PBS per well for cells from a 6-well plate
3.3 Pellet the cells by centrifugation at 400 × g for 5 minutes. Discard the supernatant.
3.4 Repeat the PBS wash 2 more times for a total of 3 washes to remove serum.
3.5 Pellet the cells by centrifugation at 400 × g for 5 minutes. Discard the supernatant. The cell pellet can be used directly in step or flash frozen and stored at –80°C until use.
3.6 Proceed to the cells lysing step.
Harvesting Adherent Cells
Note: If analyzing cell surface proteins, do not use trypsin to detach cells, because trypsin cleaves cell surface proteins. You can lyse the cells directly in the culture dish, or, if desired, use a nonenzymatic dissociation buffer or a cell scraper, and pellet the cells. You can use the cell pellet directly in next step or flash freeze and store it at –80°C until use.
4.1 Remove the medium and wash the cells three times with PBS.
4.2 Proceed immediately to the cells lysing step.
Harvesting Suspension Cells
Do not use DTT, TCEP, or β-mercaptoethanol because they will reduce the azide.
5.1 Prepare the lysis buffer by adding protease and phosphatase inhibitors at appropriate concentrations to 1% SDS in 50 mM Tris-HCl, pH 8.0. You need 500 μL lysis buffer per 100 mm dish and 200 μL lysis buffer per well of a 6-well plate.
Note: Protease and phosphatase inhibitors are optional but recommended to ensure sample integrity. If a probe sonicator is not available, add 250 U of Benzonase? endonuclease per mL of lysis buffer.
5.2 For adherent cells, add 500 μL lysis buffer per 100 mm plate or 200 μL lysis buffer per well of a 6- well plate to the labeled cells. If adding the lysis buffer directly to the plate, tap or rotate the plates so the lysis buffer covers the bottom surface of the plate. For suspension cell pellet, add 50 μL lysis buffer per 1 × 106 cells.
5.3 Incubate the cells for 15–30 minutes on ice, then tilt the plates and pipet the lysate into a 1.5 mL microcentrifuge tube. If the lysis buffer does not contain Benzonase? endonuclease, the lysate may be very viscous due to the DNA from the lysed cells. If using Benzonase? endonuclease, proceed to step 5.5
5.4 Sonicate the lysate with a probe sonicator to solubilize the proteins and disperse the DNA.
5.5 Vortex the lysate for 5 minutes.
5.6 Centrifuge the cell lysate at 13,000–18,000 × g at 4°C for 5 minutes.
5.7 Transfer the supernatant to a clean tube and determine the protein concentration using BCA assay or another method.
5.8 The protein sample is now ready for reaction with an azide or alkyne detection molecule. If not used immediately, store the sample at –20°C for up to 2 weeks. If you wish to store the sample for more than 2 weeks, precipitate the sample.
References
1. Chang, P. V., et al, (2009). Metabolic Labeling of Sialic Acids in Living Animals with Alkynyl Sugars. Angew. Chem. Int. Ed. 48, 4040-3.
2. Laughlin S.T., et al, (2007). Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation. Nature Protocols., 2, 2930–44.
