In-Solution Digestion
PDF file for
in-solution digestion protocol
Chemical Reagents: See
in-gel digestion
Solutions:
- 200 mM Dithiothreitol (DTT)
30.86 mg of DTT +1000
mL
of 100mM NH4HCO3
- 50 mM NH4HCO3:
100
mL
of 1 M NH4HCO3in 900
mL
of
ddH2O
- 1M iodoacetamide in 100 mM NH4HCO3:
Add 37 mg of iodoacetamide
to 200
mL
of 100mM NH4HCO3
- Buffer A: 50% Acetonitrile
(ACN) and 0.1% formic acid in ddH2O
25 mL of ACN + 50
mL
of formic acid + 25 mL ddH2O
- Buffer B: 0.1% formic acid
in ddH2O
1. Sample Preparation
Lower sample volumes/higher protein concentrations work best here.
Bring samples up to 100
mL
in 50mM NH4HCO3.
It is very important to adjust the pH to the appropriate pH for
certain enzyme (see Table 2 in
in-gel digestion).
It is highly recommended that the samples be free of any detergents
before digestion (most detergents can be removed by protein
precipitation and/or ion exchange chromatography see separate
protocols).
2. Disulfide Reduction
Reduce the sample by adding 5mL
of DTT stock to the 100 mL sample and
boil it for ten minutes if there is no urea in it,
otherwise skip boiling and vortex the sample, spin it down with a
quick burst in the centrifuge and let the sample reduce at room temp
for 45min-1hr.
3. Sulfhydryl Alkylation
Alkylate the sample by adding 4
mL
of the iodoacetamide stock to the sample and vortex followed by a
quick spin to get the sample to the bottom of the tube. Alkylate for
45min-1hr at room temp.
4. Stopping Alkylation
Neutralize the remaining iodoacetamide by adding 20
mL
of your DTT stock, vortex, spin, and incubate at room temp for
45min-1hr.
5. Trypsin Digest
|
1 |
The ratio of trypsin to sample should between 1:50 to1:20 1 mg
of trypsin for every 50 to 20 mg
of protein.
In order for the trypsin to work the concentration of
detergents (ie. SDS) and other chaotropes (ie. Urea) need to be
at a compatible final concentration.
When you add the correct amount of trypsin make sure you add
it in a volume that will dilute your sample to the desired
concentration of the limiting interfering agent.
See table at the end of this section for other reagent
concentrations compatibilities of trypsin. |
| 2 |
Gently vortex and spin the sample. To allow
complete digestion place the rack in the 37°C incubator
overnight, or for at least 18hrs. Gentle or periodic mixing is
optional. |
List of common reagents used for protein extraction and the
limitations of their FINAL concentrations for maintaining the enzyme
activity of trypsin.
| |
Maximum allowable concentration |
| Chaotropes: |
Less than 1
M total |
| Urea |
Thiourea
|
|
Detergents: |
| SDS |
0.05 % |
| Triton X-100 |
1 % |
| CHAPS |
1 % |
| NP-40 |
1 % |
| Tween 20 |
1 % |
| Octyl glucopyranoside
|
1 % |
|
Salts/Buffers/pH: |
Less than 250 mM total pH
~ 8-9
|
|
Reducing Agents:
|
| DTT |
20 mM |
| TCEP |
5 mM |
| TBP |
5 mM
|
| Protease
Inhibitors: |
Digestion should be free
of ALL serine protease inhibitors
|
|
Solvents: |
| Acetonitrile |
40 % (v/v) |
6 Sample Clean-up
If detergents were included in the digest they will have to be
removed first. For non-ionic detergents (ie. Tween, NP-40, CHAPS,
Triton-X), or anionic (ie. SDS), strong cation exchange (SCX)
chromatography is the recommended removal method.
Sample De-Salting and Concentration by SPE C18
Reverse Phase
This allows for the concentration of the digest and removal of
hydrophilic buffer components prior to MS analysis.
|
1 |
It is recommended that an SPE cartridge with
~100mg of resin and a volume of at least 1 mL is used. (ie. JT
Baker 10-SPE 1mL C18 extraction columns #7020-1
or
equivalent). |
| 2 |
To activate the C18 column in
order to bind your peptides wash the column with 3 X1mL of
Buffer A, then 3 X 1mL Buffer B, sending the flow through to the
waste beaker. |
| 3 |
Acidify your sample to 0.2% formic acid and
pass it over the SPE cartridge twice. The peptide-depleted
sample that contains the unbound material should be left in the
sample tube just in case. |
| 4 |
Wash unbound components off the column with 3
X 1mL of Buffer B. |
| 5 |
Elute the peptides off of the column with
400 mL
of Buffer A into a 0.5 mL tube. |
| 6 |
Reduce the sample volume and remove the
acetonitrile by vacuum concentration. |
TOP |
