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In-gel digestion of Coomassie stained bands with reduction and alkylation
- Add 100 uL of 100 mM amm bic. to gel (or enough liquid to cover the gel). Leave for 5-10 minutes.
- Remove buffer with pipette and add 50 uL of 1:1(v/v) 50mM amm bic/ACN to gel pieces. Leave for 5-10 minutes.
- Repeat steps 1 and 2.
- If gel pieces are still blue, repeat steps 1 and 2
Reduction and Alkylation
- Add 25uL of 50 mM amm bic to gel pieces.
- Add 50 uL of 10mM DTT (made up in 100mM amm bic - make up FRESH).
- Incubate at 50°C for 30 min. If gel pieces are large, use more volume but keep the ratio of 10mM DTT to amm bic the same.
- Remove DTT solution
- Add 25 uL of 50 mM amm bic to gel pieces.
- Add 50 uL of 55mM iodoacetamide (made up in 100mM amm bic - make up FRESH and keep in the dark).
- Incubate at room temperature in the dark for 20 min.
- Remove iodoacetamide solution.
- Wash with 100 uL of 50mM amm bic. Leave for 5 min, then remove.
- Wash with 100 uL of ACN. Leave for 5 min, then remove.
- Repeat this step with occasional vortexing, until gel pieces become opaque white and completely dehydrated, then remove acetonitrile.
- If you have speed vac dry the samples in speedvac for 2-3 minutes to completely evaporate acetonitrile.
- Add 25 ul of 6ng/ul trypsin to cover all gel pieces. Leave it for 15-20 minutes and observe that majority of trypsin has been absorbed by the gel pieces, remove extra trypsin.
- Add 75 uL of 50mM amm bic to keep gel pieces hydrated during the digest.
- Incubate overnight at 37°C.
- Remove supernatant - KEEP
- Add 100 uL of 1% formic acid/2% ACN to gel pieces. Leave for 5 min, then transfer to vial containing supernatant.
- Add 100 uL of 1:1 ACN/water. Leave for 5 min, then transfer to vial containing supernatant.
- Add 100 uL of 1% formic acid in ACN. Leave for 5 min, then transfer to vial containing supernatant.
- Dry extracted peptides.
- Desalt extracted peptides using zip-tip or stage-tip.
In-Solution Reduction/Alkylation Digestion
Reduction and Alkylation
- Lyophilize protein sample (10-20 µg).
- Dissolve protein pellet in 40 µL 8 M Urea (0.48 g of fresh urea dissolved in 100 mM Tris-HCl at pH 8.5 to a total volume of 1 mL) (denatures proteins).
- Add 1.25 µL of 100 mM TCEP (Tris(2-CarboxyEthyl) Phosphine) in LC-MS water (reduces disulfides).
- Incubate at room temperature for 20 minutes.
- Add 1.8 µL of 250 mM iodoactamide (prepared fresh in LC-MS water - 4.6 mg / 100 µL) and incubate for 15 minutes at room temperature in the dark (alkylates cysteines to prevent disulfide bond formation and their effect on peptide sequencing).
- Add 1 µL of 0.1 µg/µL of Lysyl endopeptidase (LysC, which is from Achromobacter lyticus with cleavage occurring at lysine and is more stable than Trypsin under high urea denaturing conditions).
- Incubate for 4 hrs. at room temperature in the dark.
- Dilute sample to final concentration of 2 M urea by adding 120 µL of 100 mM Tris-HCl pH 8.5 (dilutes denaturant to a suitable concentration for trypsin activity).
- Adjust to 1 mM CaCl2 by adding 1.6 µL of 100 mM CaCl2 (enhances trypsin activity).
- Add 2.5 µL of 0.2 µg/µL trypsin in water (trypsin cuts the protein backbone after Arg/Lys from C- to N- terminus).
- Incubate in the dark overnight at room temperature.
- Add 8.5 µL pure formic acid to bring to a final 5% formic acid concentration (inhibits trypsin and protonates tryptic peptides (charged for electrospray)).
- Desalt mixture by HPLC with a Microm Bioresources C8 peptide Microtrap (for up to 20 µg) or Macrotrap (for 20-200 µg), and lyophilize (alternatively, desalt by ZipTip for up to 5 µg or by StageTip for up to 20 µg).
- Leave lyophilized peptides dry until ready for analysis then resuspend lyophilized peptides in 0.2% formic acid at 1 µg/µL. Take 2 µL and dilute into 18 µL 0.2% formic acid for a 0.1 µg/µL final concentration. Transfer into autosampler vial, label with sample name and date, and give to PEL staff. Save remaining 1 µg/µL sample in freezer as backup.