In-gel digestion of Coomassie stained bands with reduction and alkylation
De-staining
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.
Trypsin Digestion
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.
Extraction
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).
Trypsin Digestion
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)).
Extraction
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.
