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In-gel digestion for mass spec

Author: Tie-Mei Li, updated date: , view: 194, Q&A: 0

Procedure

Stain protein gel: For silver staining, follow the basic stain protocol in SilverQuest™ Silver Staining Kit (LC6070). For coomassie staining, avoid methanol in all buffers when the purpose is to identify methylation sites.

Cut gel: cut the target gel piece into 1 mm3 pieces on a clean glass plate of plastic sheet. Put the gel pieces in a 1.5 mL tube.

Destaining after silver stain: Follow the destaining procotol described in SilverQuest™ Silver Staining Kit.  

Destaining after coomassia stain: destain the gel pieces with 1mL 50% acetonitrile at 37°C,  10min x 3 times or until no blue color. Wash with 1mL water for 10min x 3 times.

In-gel Digestion:

1. Destain the gel and wash thoroughly with water as described above.

2. Add 1 mL DTT (10 mM), 56 °C for 40 min.

3. Remove DTT, add 50 mM IAA to cover the gel slices (make 500 mM stock fresh each time: dissolve 46.4 mg Iodoacetamide in 0.5 mL 50 mM NH4HCO3 ). React at room temperature for 1 hr in the dark.

4. Wash twice with 1 mL water, 20 min each time.

5. Add 1 mL acetonitrile to make the gel shrink. Mix by turning the tube upside down for several times. Remove the acetonitrile and repeat once (until the gel turn white).

6. Speedvac for 5 min to remove the remaining acetonitrile.

7. Prepare 10 ng/uL trypsin in 50 mM NH4HCO3. Add 100-200 ng trypsin to each sample (usually make trypsin:protein = 1:20 – 1:50 by weight, if don’t know the amount of the proteins, use 200 ng trypsin)

8. Incubate at 37 °C for 15 min. Check if the liquid can cover the gel slides. If not, add 50 mM NH4HCO3 until the buffer cover the gel slides. Digest at 37 °C overnight.

9. Add 30-50 uL Extraction Buffer I (50% acetonitrile, 1% formic acid in water) to extract peptides from the gel. Rotate at room temperature for 30-60 min. Transfer the liquid to a fresh tube.

10. Add 30-50 uL Extraction Buffer II (75% acetonitrile, 1% formic acid in water) to the gel slides and rotate for 30-60 min. Combine the liquid with the one from step 9.

11. Specdvac to remove acetonitrile (or dry the sample for long-term storage). 

12. Continue to Stagetip.

 

Stage tip

  1. prepare      C18 stagetips (we use the Thermo Scientific SP301)

  2.  load stage tips with:1x 50uL MeOH, spin      at 3,000 rpm for 1 min (use this speed in the following steps unless noted      otherwise)*

  3. 50      uL Buffer B (= elution buffer)*

  4. 2x      50 uL Buffer A(=wash buffer)*

  5. Acidify      samples with 5 uL 10% formic acid, or until pH<3.

  6. Load      samples on stage dips

Spin at 2,000 rpm, 5 min (slower speed so the peptides can bind to C18)*

  1. Wash      1x 50uL buffer A*

*important note: make sure all liquid is gone before you load next step

  1. Elute      with 30 uL Buffer B , spin at 2,000 rpm, 5 min (slower speed to elute      peptides)

  2. Speedvac      plate until dry, store at -20 degree

 

For LC-MS:

  1. Resuspend      the peptides in 10 uL (2% acetonitrile, 0.1% formic acid in water), vortex

  2. Spin      10 min at max speed to remove any pellet.

  3. Transfer      to sample vial. Send to Elias lab.

 

Buffer A: 0.1% formic acid in water

Buffer B: 60% acetonitrile, 0.1% formic acid in water


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