In-gel Digest Protocol: ProteaseMax and Overnight

Saveliev et al. Enhanced and rapid in-gel protein digestion with a mass spectrometry
compatible surfactant. 2012.

Proteomics Core in-gel digested protocol modified from Shevchenko and Children Hospital’s
digest protocol. The digestion step as well as extraction is following Saveliev et al’s protocol.

IN-GEL REDUCTION/ALKYLATION

● Remove gel from Eppendorf tube and cut band into ~1mm​3​ pieces—place gel pieces
back into the tube
● Wash the gel pieces with 100-150 ul of 50mM ammonium bicarbonate (AMBIC). Repeat
this step 1-2x
○ Volume of AMBIC is arbitrary—add enough to cover entire gel piece
● Add acetonitrile (same volume as was used in the AMBIC step) and agitate until the gel
pieces shrink, become opaque, and stick together. Repeat this step 2-3x.
● Swell the gel pieces in 10 mM dithiothreitol (DTT) in 50 mM AMBIC and incubate for 30
minutes at 56°C to reduce the disulfide bonds in the protein.
● Shrink the gel pieces with acetonitrile. Repeat this step 2-3x
● Replace the acetonitrile with 55 mM iodoacetamide (IAA) in 50 mM ammonium
bicarbonate. Incubate for 20 min at room temperature in the dark.
○ IAA is light sensitive—store in a dark box until ready to use
● Discard iodoacetamide solution. Wash the gel pieces with 150-200uL of 50 mM
ammonium bicarbonate (1-2x).
● Shrink the gel pieces with acetonitrile (1-2x)
● Dry in the speed vac until gel pieces are completely dry and not cold to the touch.
Alternatively, do additional acetonitrile washes

Protease Max Digestion

● Add 100uL of 50mM AMBIC (pH 8) to a vial of ​Protease Max​ to get a 1% solution
● Make a solution of 0.01% Protease Max by adding 10uL of 1% ProteaseMax solution to
990uL of 50mM AMBIC
● Add ​Promega trypsin ​in a 1:30 ratio (enzyme:protein) and just enough of the 0.01%
Protease Max solution to cover the gel (30-50uL depending on the size of the gel)
● Let sit at room temperature for 10 minutes
● Add 50-100uL of 0.01% Protease Max and incubate at 50° C for 1-4 hours
● After incubation, add 5uL formic acid, centrifuge for 5 minutes, and collect the
supernatant
Alternate Digestion: Overnight

● Place trypsin (1:30 ratio, enzyme:protein) on the side of the eppendorf test tube, not
touching the gel pieces and then resuspend the gel pieces as well as the trypsin in
50mM AMBIC. Make sure that the AMBIC has a pH of 8 prior to addition of the trypsin.
Add enough digestion buffer so that after the gel pieces absorb the buffer, they will still
be completely covered in AMBIC
● Digest overnight​ ​@37°C

Extraction

● Collect the supernatant and place it in a fresh Eppendorf test tube.

● Add just enough 60% acetonitrile and 0.1% trifluoroacetic acid (TFA) to cover the gel
pieces and sonicate in the water bath for ten minutes.
● Centrifuge for 10 minutes.
● Collect the supernatant and add it to the supernatant collected earlier.
● Dry down the mixture in the speed vac, but stop before completely dried( about 10uL of
liquid left)