Sample preparation for Mass analysis

Preparation of chemicals

Chemicals
Methanol (MeOH) for blotting sequence type
Acetonitrile CH3CN), HPLC grade
Trifluroacetic acid (TFA) HPLC grade 

Stock solutions
(Preserve at 4 ºC)
100 mM NH4HCO3
500 mM EDTA.2Na

Washing solution (25% MeOH / 7% Acetic acid)
10 mM Tris-HCl  (pH 8.0)
3% TFA
0.1%  TFA / 100% CH3CN
0.1% TFA / 100% H2O
Freezer (-20 ºC)
1 M Dithiothreitol (DTT)
10 pM Trypsin / 10 mM Tris-HCl

Chemicals to be prepared during use
Destaining solution (50 mM NH4HCO3 / 50% MeOH)
100 mM NH4HCO3                    2.5 ml
100% MeOH                         2.5 ml

Reduction solution ( 10 mM EDTA.2Na / 10 mM DDT / 100 mM NH4HCO3
500 mM EDTA.2Na                    20 µl
1M DDT                             10 µl
100 mM NH4HCO3                    970 µl

Alkylation solution (10 mM EDTA. 2Na / 40 mM Iodoacetamide / 100 mM NH4HCO3
500 mM EDTA.2Na                    20 µl
Iodoacetamide                     7.4 mg
100 mM NH4HCO3                    980 µl

Trypsin solution
10 pM Trypsin / 10 mM Tris-HCl     50 µl
10 mM Tris-HCl (pH 8.0)           450 µl


Sample preparation
1. Cut the gel (spot)
Cut the selected protein spots from the 2D-gels.
Place the gels on light box and cut the protein spots with a spatula. 
Collect the gel pieces in an eppendorf tube. 
Wash the gel pieces with water (MQ), drain out water. 
This can be preserved in freezer at -20 ºC.  
Or spot picker (Fig.3)

2. Washing and destaining
Add 1 ml wash solution (25% MeOH / 7% Acetic acid) to the eppendorf tube, 
fix them in a rotor and rotate over night at room temperature.

Destaining procedure depends on the method of staining:

In case of CBB staining: Wash with water (MQ), add 0.5 ml destaining solution (50 mM NH4HCO3 / 50% MeOH), 
incubate at 40 ºC until the samples (gel pieces) are destained (may take 30 to 60 min). 
After destaining, dry out destaining solution then wash with water (MQ).
In case of silver staining:  Add 0.3 ml solution (20 mM EDTA. 2Na / 50 mM Tris-Hcl (pH8.0) / 30% CH3CN), 
wait for 5 min at room temperature. Drain out supernatant, repeat this step once more. 
Then wash with water (MQ) for two times.

3. Reduction and Alkylation
1) Dry the samples in speed Vac. concentrator (may skip this step)
2) Add 50 µl reduction solution (10 mM EDTA.2Na / 10 mM DTT / 100 mM NH4HCO3) 
             to the eppendorf tube to make the gels wet. Incubate for 1 h at 60 ºC placing on a cool block.
3) Discard the solution and dry again using speed Vac.
4) Add 50 l alkylation solution (10 mM EDTA.2Na / 40 mM Iodoacetamide / 100 mM NH4HCO3) 
   to the gels in eppendorf tube, place in dark at room temterature for 30 min to carry out reaction.
5) Add 1 ml water (MQ), wash the gels and drain the water. Repeat this step for 2 times. 
6) Smear the gels with a Pelletier. 
7) Dry the gels in a speed Vac. concentrator.

4. Digestion
1) Prepare 1 pmol trypsin solution (10 pM Trypsin / 10 mM Tris-HCl), add 50 µl to the gels in eppendorf tube.  
2) Wrap the eppendorf tube with Para film, digest at 37 ºC for 10 to 12 h.

5. Elution
1) Add 100 µl solution (0.1% TFA / 100% CH3CN) to the gels in eppendorf tube(A), sonicate for 10 min.
2) Collect the supernatant in a new eppendorf tube (A1).
3) Add 100 µl solution (0.1% TFA / 100% MQ) to the gels in eppendorf tube (A), sonicate for 10 min. 
4) Collect the supernatant in eppendorf tube (A1).
5) Add 80 µl solution (0.1% TFA / 50% CH3CN) in the gels in eppendorf tube (A), sonicate for 10 min.  
6) Collect the supernatant in eppendorf tube (A1).
7) Add 100 100 µl solution (0.1% TFA / 100% CH3CN) to the gels in eppendorf tube(A), sonicate for 10 min.
8) Collect the supernatant in a new eppendorf tube (A1).
9) Concentrate the collected sample to 20 - 30 µl in a speed Vac. (CH3CN will be evaporated). 
     This sample is peptide solution.
     Or Robots. (Fig.4)

6. Desalt
1) Using reverse phase column, desalt and concentrate the peptide solution. 
2) Wash with ZipTip C18 (Millipore) filling and unfilling 10 µl 0.1% TFA / 50% CH3CN 3 times
3) Wash and stabilize ZipTip with 10 µl 0.1% TFA / 100% MQ, filling and unfilling 3 times 
4) Take the whole peptide solution in ZipTip (10 µl each time) in repeated times, 
   thus absorbing the whole peptides in ZipTip columniv) 
   Wash and desalt the ZipTip with 10 µl 0.1% TFA / 100% MQ, repeat 3 times
5) Dissolve the peptides absorved in column by 0.1% TFA / 50% CH3CN. 
   The sample volume depends on the following operation. 

 In case of analyzing using VOYAGER-RP:
 Use 2 µl peptide on the sample plate and 2 µl Matrix solution, mix them well, air dry and analyze. 

 In case of analyzing using other Mass spectrometer
 The sample volume depends on requirement.

 In case of preservation for future analysis
 Take 5 in an eppendorf tube and keep in freezer at -20ºC.
 (Fig.5-1, Fig.5-2), 

Matrix Preparation

1. Selection of Matrix
Select Matrix based on the molecular size of the sample
  > 10 kDa              CHCA (α-Cyano-4-Hydroxycinnamic Acid)
  < 10 kDa              SA (Sinapinic Acid)

2. Preparation
Prepare just before use. Mix the following chemicals using vortex for 1 min, 
leave it for 10 min, use the supernatant (not completely separated supernatant).

      CHCA  or  SA            10 mg
      3% TFA                 100 µl
      CH3CH                  500 µl
      H2O                    400 µl