TITLE: Gel Slice Sample Preparation for Proteomics
AUTHORS: Jennifer Gin, Leanne Chan, Christopher Petzold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details steps for gel slice sample preparation for proteomic data acquisition. It was adapted from Shevchenko et al. "In-gel digestion for mass spectrometric characterization of proteins and proteomes."</span><span style = "font-style:italic;">Nature protocols </span><span>1.6 (2006): 2856.</span></div></div>

[STEPS]
?. [Day 1:  Washing the gel slices]
Wash gel slice with in a tube (Eppendorf Centrifuge, Cat.#022363204). Incubate for  then remove liquid.
[Water]
?. [Day 1:  Washing the gel slices]
Wash gel slice withand incubate for  then remove liquid.
[50 mM AMBIC/ACN]
Discard used buffers and solvents in appropriate waste containers.
?. [Day 1:  Washing the gel slices]
Add  and incubate for  then remove liquid.
[50 mM AMBIC/ACN]
?. [Day 1:  Washing the gel slices]
Add to cover gel slice. The gel slice will visibly shrink and turn white. Incubate for  to  then remove liquid.
[Acetonitrile]
?. [Day 1:  Trypsin digestion]
Add  and incubate in  for  then remove liquid.
[10 mM DTT, 25 mM AMBIC]
[water bath]
?. [Day 1:  Trypsin digestion]
Add  and incubate in the dark, at   for  then remove liquid.
[55 mM IAA, 25 mM AMBIC]
0 Room temperature
?. [Day 1:  Trypsin digestion]
Wash gel slice by addingand incubating for then remove liquid.
[50 mM AMBIC/ ACN]
?. [Day 1:  Trypsin digestion]
Repeat wash step 9.
?. [Day 1:  Trypsin digestion]
Add to cover gel slice. The gel slice will visibly shrink and turn white. Incubate for  to  then remove liquid..
[Acetonitrile]
?. [Day 1:  Trypsin digestion]
Add and incubate on  for  , then remove excess liquid, if necessary.
[12.5 ng/ml Trypsin, 50 mM AMBIC]
on ice
All of the liquid (trypsin) should be absorbed into the gel slice at the end of 30 minutes. The gel slice will look clear/hydrated again. There is usually no liquid leftover because only a small amount of trypsin is added however, if necessary, remove excess trypsin before overnight digestion.
?. [Day 1:  Trypsin digestion]
Add  to cover the gel slice.
[AMBIC]
Wait 15-20 minutes to make sure the gel slice is still completely covered in liquid. Sometimes the gel is not completely rehydrated after the trypsin has been absorbed, and it absorbs a lot of the AMBIC if you only use enough to cover. This is especially a problem with larger gel pieces.
?. [Day 1:  Trypsin digestion]
Digest the protein(s) at  in an incubator or thermocycler.
37 °C
?. [Day 2:  Recovering digested peptides]
Remove gel slice from  incubator or thermocycler.
37 °C
?. [Day 2:  Recovering digested peptides]
Add   after tryptic digestion. Incubate at  for  .
[50 mM AMBIC]
0 Room temperature
?. [Day 2:  Recovering digested peptides]
Add to cover the gel slice.
[50% ACN/ 5% FA]
?. [Day 2:  Recovering digested peptides]
Incubate for   on a benchtop shaker (Pipette.com, Cat.#2231000667) set at  .
0 Room temperature
?. [Day 2:  Recovering digested peptides]
Collect liquid.
?. [Day 2:  Recovering digested peptides]
Repeat steps 18-20.
?. [Day 2:  Recovering digested peptides]
Add . Incubate for then collect liquid.
[Acetonitrile]
?. [Day 2:  Recovering digested peptides]
Use a SpeedVac (Fisher Scientific, Cat.#07-748-13) to dry the liquid completely.
?. [Day 1:  Washing the gel slices]
Add  and incubate for  then remove liquid.
[ Acetonitrile]
?. [Day 2:  Recovering digested peptides]
Collect liquid in a clean tube.
?. [Buffer Preparation]
Buffers to prepare fresh:  AB1100 ml of 100 mM Ammonium Bicarbonate (AMBIC):0.79 g Ammonium Bicarbonate (VWR Scientific, Cat.#BJ40867-50G)    
100  ml LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5)21 ml of 50mM Ammonium Bicarbonate/Acetonitrile (AMBIC/ACN):250 µl 100 mM Ammonium Bicarbonate                                                    
250 µl LC-MS grade Water                                                                                                       
                   500 µl Acetonitrile (VWR Scientific, Cat.#BJLC015-2.5)31 ml of 10 mM 1,4-Dithiothreitol (DTT), 25 mM Ammonium Bicarbonate (AMBIC):10 µl 1M 1,4-Dithiothreitol (Millipore Sigma, Cat.#DTT-RO)                       
250 µl 100 mM Ammonium Bicarbonate                                                      
740 µl LC-MS grade Water41 ml of 55 mM Iodoacetamide, 25 mM Ammonium Bicarbonate (IAA/AMBIC):10 mg Iodoacetamide (Millipore Sigma, Cat.#I1149)                                 
     250 µl 100 mM Ammonium Bicarbonate                                                      
750 µl LC-MS grade Water50.200 ml of 100 ng/ul of Trypsin:20 µg Sequencing Grade Modified Trypsin (Promega, Cat.#V511A)       
200 µl 50 mM Acetic Acid Millipore Sigma, Cat.#818755)60.100 ml of 12.5 ng/ml Trypsin/50 mM Ammonium Bicarbonate (Trypsin/AMBIC):12.5 µl 100 ng/ul Trypsin                                                                                    
     50 µl 100 mM Ammonium Bicarbonate                                                       
37.5 µl LC-MS grade Water71 mL of 50% Acetonitrile/5% Formic Acid (ACN/FA):0.5 ml Acetonitrile                                                                                            
0.05 ml Formic Acid (Thermo Fisher Scientific, Cat.#28905)                   
  0.45 ml LC-MS grade Water81mL of Buffer A0.001 ml Formic Acid                                                                                          
     1 ml Water
AB1100 ml of 100 mM Ammonium Bicarbonate (AMBIC):0.79 g Ammonium Bicarbonate (VWR Scientific, Cat.#BJ40867-50G)    
100  ml LC-MS grade Water (VWR Scientific, Cat.#BJLC365-2.5)21 ml of 50mM Ammonium Bicarbonate/Acetonitrile (AMBIC/ACN):250 µl 100 mM Ammonium Bicarbonate                                                    
250 µl LC-MS grade Water                                                                                                       
                   500 µl Acetonitrile (VWR Scientific, Cat.#BJLC015-2.5)31 ml of 10 mM 1,4-Dithiothreitol (DTT), 25 mM Ammonium Bicarbonate (AMBIC):10 µl 1M 1,4-Dithiothreitol (Millipore Sigma, Cat.#DTT-RO)                       
250 µl 100 mM Ammonium Bicarbonate                                                      
740 µl LC-MS grade Water41 ml of 55 mM Iodoacetamide, 25 mM Ammonium Bicarbonate (IAA/AMBIC):10 mg Iodoacetamide (Millipore Sigma, Cat.#I1149)                                 
     250 µl 100 mM Ammonium Bicarbonate                                                      
750 µl LC-MS grade Water50.200 ml of 100 ng/ul of Trypsin:20 µg Sequencing Grade Modified Trypsin (Promega, Cat.#V511A)       
200 µl 50 mM Acetic Acid Millipore Sigma, Cat.#818755)60.100 ml of 12.5 ng/ml Trypsin/50 mM Ammonium Bicarbonate (Trypsin/AMBIC):12.5 µl 100 ng/ul Trypsin                                                                                    
     50 µl 100 mM Ammonium Bicarbonate                                                       
37.5 µl LC-MS grade Water71 mL of 50% Acetonitrile/5% Formic Acid (ACN/FA):0.5 ml Acetonitrile                                                                                            
0.05 ml Formic Acid (Thermo Fisher Scientific, Cat.#28905)                   
  0.45 ml LC-MS grade Water81mL of Buffer A0.001 ml Formic Acid                                                                                          
     1 ml Water
Store DTT in   and Trypsin in  .IAA is light sensitive.  Store in amber tube (Fisher Scientific, Cat.#05-402-31).
?. [Day 2:  Sample Prep For MS]
After the tubes are completely dry in Step 24, add   then transfer to plastic autosampler vials (Agilent, Cat.#5182-0567,#5182-0564) or 96-well plate (Bio-Rad, Cat.#HSP9655).
[Buffer A]
?. [Day 2:  Sample Prep For MS]
Store at   until ready for LC-MS/MS analysis.
-20 °C