TITLE: Reduction and alkylation of protein lysates for LC-MS (proteomics) using dithiothreitol (DTT) and iodoacetamide (IAM)
AUTHORS: ronan.ocualain

[DESCRIPTION]
This protocol details the procedure of the reduction and alkylation using dithiothreitol (DTT) and iodoacetamide (IAM).

[BEFORE_START]
 Initial preparation:

Before you begin:

Locate the Eppendorf Thermomixer, and attach the appropriate heating block depending on whether your samples are in 0.5 mL or 1.5 mL tubes. Set the temperature to 60 °C and a speed of 500 rpm.



[GUIDELINES]
Reduction and alkylation of cysteine bonds is a prerequisite for LC-MS sample preparation for two reasons:
Cysteine bonds are part of the proteins secondary structure. By reducing (breaking) them, it allows better access of trypsin or other digestion enzymes for the complete conversion of protein to peptide.
It is not straightforward to identify cysteine containing peptides using LC-MS, because they usually are two peptides, linked by at least one cysteine bond. This will not be matched when the data is searched against the database, and will limit protein sequence coverage in the results.
It is a sequential reaction. Dithiothreitol (DTT) is used to reduce the cysteine bonds present in the protein. After a short incubation, iodoacetamide (IAM) is added to modify the free cysteines. After another short incubation, DTT is added again to quench any free IAM.

Initial assumptions and preparation:

Allow approximately 90 min reduction and alkylation.
You have protein lysates in Eppendorf tubes in a known volume of S-Trap lysis buffer.

[STEPS]
SECTION: Before you begin
2. Remove the DTT and IAM aliquots from fridge 2.
 



SECTION: Before you begin
4. Take one of each for your preparation. Place the boxes back in the fridge. 
SECTION: Before you begin
5. To make a 100 millimolar (mM) solution of DTT and IAM, add the volume of water indicated on the box from which you took the pre-weighed aliquot.

 

SECTION: Reduction and alkylation:
6. You will need to know the volume of the protein lysate sample that you wish to alkylate. If proceeding from LE220+ sample processing, this will be approximately 130 uL for the smaller tubes, and 500 uL for the larger tubes.
SECTION: Reduction and alkylation:
7. Reduce your protein sample.
 

SECTION: Reduction and alkylation:
8. For the 500 µL lysate, add 25 µL of the stock 100 millimolar (mM) DTT.
SECTION: Reduction and alkylation:
9. Place the tubes on the Eppendorf thermomixer and heat for 10 min at 60 °C.
 

SECTION: Reduction and alkylation:
10. Alkylate your protein sample. Remove the protein samples from the thermomixer, and allow to cool to Room temperature. Add IAM. It is important to add at least three times the amount of IAM to DTT. So alkylate with 15 millimolar (mM) of IAM.
SECTION: Reduction and alkylation:
10.1. For 130 µL of protein sample, add (15 * 130) / 100 or 20 µL of IAM to the samples, and for 500 µLof sample, add (15 * 500) / 100  or 75 µL of IAM to the samples.
SECTION: Reduction and alkylation:
11. Vortex mix the tubes briefly and place the protein sample tubes in the dark (a drawer) for 30 min.
SECTION: Reduction and alkylation:
12. After 30 min, add DTT to quench the alkylation reaction. Add the same amount of DTT again as to what you added in the reduction step. Vortex mix briefly.

 


SECTION: Reduction and alkylation:
13. Centrifuge your samples at 14000 rcf for 10 min using the centrifuge in room B2075.
To use, switch is located at rear of machine. To swop rotors, press down on the green spindle, and lift the rotor upwards. The centrifuge is for using a plate rotors, and a tube rotor. The rotors are located to the right of the machine, along with balances. 
Tubes and plates must be balanced!
 

SECTION: Reduction and alkylation:
14. Remove the supernatant using a clean pipette tip to a labelled tube. This is the protein lysate.
SECTION: Reduction and alkylation:
15. You have now reduced and alkylated your protein lysate samples.
 

SECTION: Reduction and alkylation:
7.1. If the starting concentration (Sc) of the stock DTT is 100 millimolar (mM), and the volume of sample (Fv) to be reduced is 130 µL, then use the following calculation to work out how much of the stock DTT (Sv) to add = (Fc * Fv) / Sc.
SECTION: Reduction and alkylation:
7.2. For 130 µL of lysate, add (5 * 130) / 100, or 6.5 µL of stock 100 millimolar (mM) DTT. 
SECTION: Before you begin
3. They are pre-weighed in 1.5 mL Eppendorf tubes.

 

SECTION: Before you begin
1. Locate the Eppendorf Thermomixer, along with the thermoblock you will be using. The Thermoblock "clicks" into place onto the Thermomixer unit. Set the Thermomixer to 60 °C and a speed of 500 rpm.

 
Use the PCR 96 thermoblock for 0.75 mL Eppendorf tubes
 

Use the 1.5 mL thermoblock for 1.5 mL Eppendorf tubes.