Tryptic in-gel digestion of proteins

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					                               Tryptic in-gel digestion of proteins


1. General considerations

1.1 Working conditions

   In order to reduce the chance of keratin contamination always use nitrile gloves (change frequently),
   wear a lab coat and keep hands off face and hair while handling the gel. Also, don’t touch the gel
   with bare hands, keep the gel in solution and covered up. In order to avoid dust, i.e. keratin, work in
   an area that keeps dust to a minimum. Wipe down all surfaces in the hood with methanol/water
   moistened lint-free cloth. Wipe razor blades and tweezers with methanol-soaked lint-free cloth. Use
   Eppendorf’s LoBind tubes to increase the peptide recovery rate.

   While running a gel, include a BSA standard in the approximate concentration range of the protein
   of interest. It serves as a positive control, to ensure the overall performance of the process. Also, run
   an empty lane as a negative control sample. It is used to detect material interferences.


1.2 The serine proteinase Trypsin

   Trypsin is synthesized as trypsinogen by the vertebrate pancreas, but rapidly becomes converted by
   the removal of the amino-terminal hexapeptide to the active enzyme of approx. 23,500 Da.
   It has a serine at its active site, and therefore belongs to the serine proteinase family.
   Trypsin is considered an endopeptidase, since the cleavage occurs within the polypeptide chain
   rather than at the terminal amino acids.
   Trypsin displays good specificity, catalyzing the hydrolysis of the peptide bond to the carboxyl side
   of lysyl and arginyl residues. If an acidic residue occurs to either side of the basic residue, hydrolysis
   is slower, and if the residue to the carboxyl side is proline, hydrolysis is very slow. Trypsin is
   optimally active at about pH 8.
   Various grades of trypsin are commercially available. Most are from bovine pancreas. Enzyme
   activity may vary from source to source or batch to batch. Attention to this is recommended if
   reproducibility is important (e.g., for peptide mapping).

   During incubation with substrate, trypsin will also undergo autolysis. It will produce masses which
   can be used as internal standards, or log masses for MALDI analysis: 842,5100 ; 1045,5642 ;
   2211,1046; 2283,1807; 2299,1756 are the most important ones.
   The digestion can be terminated by immediate submission to peptide mapping or isolation
   techniques, acidification, freeze-drying, or addition of specific inhibitors. As with other serine
   proteinases, trypsin may be inactivated by reaction of the serine residue at the active site with
   phenylmethylsulfonyl fluoride (PMSF).

   For reproducible peptide mapping, when complete digestion is desirable, prolonged digestion may
   be required (e.g., 48h at 37°C, pH 8, with a second addition of trypsin, similar to the first at 24h). On
   the other hand, for preferential cleavage of particularly sensitive bonds or for production of partial
   cleavage products, digestion can be limited by use of a low enzyme/ substrate ratio, shorter digestion
   times, and lower incubation times.

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2. Experiment

     Trypsin is used to cleave proteins by hydrolyzing peptide bonds at the carboxyl side of arginyl and
     lysyl residues. Trypsin breaks up proteins separated previously by 1D- and 2D-SDS gel
     electrophoresis into peptide fragments.
     Protein bands of interest are excised, washed, reduced, alkylated and finally digested by trypsin.
     Resulting peptides are either analyzed by MALDI-TOF (with a prior Zip-Tip step for clean up and
     concentration), or by LC-MS/MS.


2.1 Equipment

o    Ice or refrigerator at 4°C
o    Aluminum foil
o    1.5mL Protein LoBind Eppendorf tubes (Eppendorf Cat.No.: 2243108-1)
o    Gel loading pipet tips
o    Glass plate, clean stainless steel razor blade
o    Heating block at 56°C
o    Water bath or heating block at 37°C
o    Speed vac with cool trap
o    Centrifuge
o    Vortexer
o    Dust free environment, i.e. hood


2.2 Chemicals

a.   Chemicals for Coomassie and Sypro Ruby stained gels

o Trypsin: Promega, Madison WI, USA (608 2744330)
  Trypsin (Porcine), Seq. Grade Modified, V511C, frozen 521 µg/ml
o Ammonium bicarbonate: Sigma, Order #: A6141-500G
o Iodoacetamide: Sigma-Ultra, Order #: I1149-5G
o DL-Dithiothreitol: Sigma, Order #: D0632-5G
o Formic Acid, 96%, A.C.S. reagent: Sigma, Order #: 251364-500G
o Acetonitrile, 99.93%, HPLC Grade: Sigma, Order #: 270717-4L
o Water, OmniSolv, High purity solvent, EMD, Order # WX0004P-1
o

b. Additional chemicals for the destaining of silver stained gels

o 30mM Fe K3(CN)6: Fluka, Order #: 60299-100G
o 100mM Na2S2O3: Aldrich, Order#: 563188-25G




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3. Destaining of gel bands

3.1 Staining methods

    The destaining method depends on the used stain. The purpose of the destaining step is to give
    trypsin an unobstructed way to the protein cleavage site.
    There are four major stains applied to SDS gels.

    1. Coomassie Blue Colloidal (protein detection limit 50 – 100ng)
    2. Coomassie Blue Brilliant (protein detection limit 100 – 150ng)
    3. Sypro Ruby (Protein detection limit below 10ng)
    4. Silver staining (Protein detection limit below 10ng)


    Proteins stained with one of the first three stains are destained using the following method:


3.2 Destaining gel pieces stained with Coomassie Blue Colloidal, Coomassie Blue Brilliant and
    Sypro Ruby

a. Prepare the following solutions:

o 25mM NH4HCO3
o 25mM NH4HCO3 in 50% ACN
o 5% formic acid in 50% ACN
o 12.5ng/ul trypsin in 25mM NH4HCO3, (add 160µL to 4µL (0.5µg/µL) aliquot) Prepare freshly, 1hr
  prior to use and keep on ice
o 10 mM DTT in 25mM NH4HCO3
o 55mM iodoacetamide in 25mM NH4HCO3
o Solution is light sensitive, so wrap the container in aluminum foil.


b. Using a razor plate cut as close as possible to the bands, dice them in 1mm2 cubes and place them
   into a 1.5mL Protein LoBind Eppendorf tube. Also, cut out and cut up a piece of unstained gel as a
   negative reference sample (preferably an empty lane).

c. Add 100µL of 25mM NH4HCO3 in 50%ACN to each sample.

d. For Coomassie Blue Colloidal/ Brilliant stained gel pieces: Vortex samples for 10 minutes, centrifuge
   gel pieces down. Check the solution; if it is blue and gel pieces are white, the destaining step was
   successful.

e. With gel loading pipet tips remove liquid.




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f. If gel pieces are still blue, rehydrate gel pieces with 100µL of 25mM NH4HCO3, vortex for 10
   minutes and centrifuge gel pieces down. Remove liquid with gel loading pipet tips and repeat steps
   c,d,e.
   For Sypro Ruby stained gels: Repeat steps c,d,e.

g. Dry gel pieces with Speed vac for about 15 minutes. Pieces look crystalline when they are dry.

h. Store gel pieces at 4°C overnight or at -20°C for longer periods of time. This is a good break point in
   the procedure.


3.3 Destaining of silver-stained bands

a. Prepare the following solutions:

o 30mM Fe K3(CN)6
o 100mM Na2S2O3
o 25mM NH4HCO3 in 50% ACN


b. Mix 30mM Fe K3(CN)6 and 100mM Na2S2O3 just prior to use in a 1:1 ratio.

c. Soak gel bands in 30µl for 10 min., vortexing in between. If necessary repeat, until gel bands are
   clear.

d. Remove the destaining solution and wash gel pieces 3 times with 100µl water, vortexing for 15 min
   for a total of 45 minutes.

e. Remove water and wash gel pieces 3 times with 100µl of 25mM NH4HCO3 in 50% ACN, vortexing
   for 15 min for a total of 45 minutes.

f. Dry gel pieces with Speed vac for about 15 minutes. Pieces look crystalline when they are dry.

g. Store gel pieces at 4°C overnight or at -20°C for longer periods of time. This is a good break point in
   the procedure.


4. Reduction and Aminocarboxymethylation of cysteine residues


    The reduction andaminocarboxymethylation of cysteine residues cause the permanent disruption of
   disulfide bonds. These treatments open the protein structure for digestion and minimize
   complications in peptide separation that are caused by pairs, or by peptides remaining connected by
   S-S bonds.

     Reduction and Aminocarboxymethylation of disulfide bonds should be included when maximum
   recovery of proteins separated by 2D gel electrophoresis is required, or when a one dimensional gel
   band is digested. Proteins without cysteines do not need to go through these steps. Remember these
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   modifications when database searches are performed (Fixed modification: Carbamidomethylation
   (C), variable modifications: Oxidation (M)).


4.1 Reduction of disulfide bonds with DL-Dithiothreitol (DTT)

   DTT is used to reduce disulfide linkages while maintaining the thiol group in the reduced state in
   solution.




a. Add 10 mM DTT in 25mM NH4HCO3 solution to dried gel pieces, so that they are just covered
   with a thin layer of liquid (25 to 35µL), vortex and spin them down.

b. Incubate gel pieces in the heating block for 1 hour at 56°C.


4.2 Aminocarboxymethylation with Iodoacetamide (IAA)

                              I-CH2-CONH2

                                         --->            -CH2-S-CH2-CONH2
       -CH2- SH + I-CH2-CONH2

       Cysteine + Iodoacetamide                 S-Carbamidomethyl cysteine



a. Remove liquid and add 25 to 35µL of iodoacetamide in 25mM NH4HCO3 to gel pieces. Vortex,
   spin and incubate them for 45 min. at RT in darkness.

b. Remove liquid with gel loading tips, discard, wash with 100µL 25mM NH4HCO3, vortex for 10
   minutes, spin gel pieces down.

c. Remove supernatant and discard, dehydrate gel pieces with 100µL of 25mM NH4HCO3 in
   50%ACN, vortex 5 minutes, spin briefly and repeat last step. Speed vac the gel pieces to complete
   dryness. Again, you can store gel pieces at 4°C overnight or at -20°C for longer periods of time.


5. Tryptic digestion

 a. Add trypsin solution to sample, so gel pieces are barely covered. Spin briefly.

 b. For rehydration put samples 10 minutes on ice.


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 c. Overlay the rehydrated gel pieces with 25mM NH4HCO3 to keep them immersed during
    digestion. Spin briefly.

 d. Incubate at 37°C from 4 to 16 hours.


6. Extraction of peptides

a. Transfer solution into clean vial 2.

b. Add 5% formic acid in 50% ACN to gel pieces so they are just covered. Vortex them for 20-30
   minutes, spin and sonicate them for 15 minutes.

c. Remove liquid and add to digest solution in vial 2.

d. Add again 5% formic acid in 50% ACN to gel pieces. Vortex 15 minutes, spin and sonicate for 15
   minutes.

e. Remove liquid and add to digest solution in vial 2.

f. Vortex, spin and reduce volume by using Speed vac. Do not completely evaporate the solvents. Keep
   approximately 10-20µL.

g. Analyze samples by Maldi-TOF or by LC-MS/MS.


7. MS-Analysis of peptides

7.1 Maldi-TOF sample analysis:

   MALDI-TOF analyses are recommended for proteins separated by 2D-gel electrophoresis, which are
   relatively pure. If MALDI-TOF analysis shows a suppressed response, a C18 Zip Tip clean up, or
   desalting, step can help to remove interference caused by the gel matrix, but it can also result in
   significant loss of peptides, if not done properly.
   A practical alternative to the Zip Tip extraction is a 1:10 dilution of the sample in 50% Acetonitrile
   with 1% TFA, if the sample is concentrated enough. It reduces background masses caused by the gel
   matrix.
   To precoat the MALDI target spot with nitrocellulose is another way to go; the sample is applied on
   top of the precoated target spot. After the sample dried, it can then be desalted with 5% Formic Acid
   in water.
   All necessary sample preparation steps will be taken by the facility.

7.2 LC-MS/MS sample analysis:

   The best way of sample preparation is the evaporization of solvent to total dryness using a speed-
   vac. The sample will be resuspended in a solvent appropriate for the analysis in the facility.



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