Aug 17, 2022
Reduction and alkylation of protein lysates for LC-MS (proteomics) using dithiothreitol (DTT) and iodoacetamide (IAM)
- 1University of Manchester
Protocol Citation: ronan o'cualain 2022. Reduction and alkylation of protein lysates for LC-MS (proteomics) using dithiothreitol (DTT) and iodoacetamide (IAM). protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr6573vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 20, 2022
Last Modified: August 17, 2022
Protocol Integer ID: 67134
Keywords: Reduction, Alkylation, Iodoacetamide, Dithiothreitol, in-solution digestion, peptide desalting proteomics, mass spectrometry, LC-MS, proteomics, Cysteine, Cystine, Disulfides, Sulfhydryl Compounds
Abstract
This protocol details the procedure of the reduction and alkylation using dithiothreitol (DTT) and iodoacetamide (IAM).
Attachments
iiaebptmp.docx
173KB
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 01:30:00 reduction and alkylation.
- You have protein lysates in Eppendorf tubes in a known volume of S-Trap lysis buffer.
Materials
Locate the following buffers, consumables, and reagents:
| A | B | |
| Location | Buffer/reagent | |
| Fridge 02 | DTT (PN# BP172-5,Fisher) pre-weighed aliquots – pink box – top shelf IAM (PN# I1149,Sigma Aldrich) pre-weighed aliquots – pink box – top shelf | |
| Bench | Eppendorf tubes, 1.5 mL volumes depending on sample volume. LC-MS grade water tinfoil | |
| Freezer | N/A |
Identify the following equipment that you will use:
- 20 µL pipette, 200 µL pipette, 1 mL pipette and pipette tips.
- Kern fine balance.
DithiothreitolFisher ScientificCatalog #BP1725
IodoacetamideMillipore SigmaCatalog #I1149
Equipment
ThermoMixer® C
NAME
Thermoblock
TYPE
Eppendorf
BRAND
5382000031
SKU
LINK
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 .
Before you begin
Before you begin
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 .
Note
PCR 96 Thermoblock
Use the PCR 96 thermoblock for 0.75 mL Eppendorf tubes
Note
1.5 mL thermoblock
Use the 1.5 mL thermoblock for 1.5 mL Eppendorf tubes.
5m
Remove the DTT and IAM aliquots from fridge 2.
Note
Fridge location, pre-weighed aliquots are on bottom shelf
5m
They are pre-weighed in 1.5 mL Eppendorf tubes.
Note
Pre-weighed aliquots
1m
Take one of each for your preparation. Place the boxes back in the fridge.
1m
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.
Note
NB. It is important to wrap the 100 millimolar (mM) solution of IAM in tinfoil when it is prepared.
Both of these solutions have a short half-life (approximately 24 hours), and must be prepared fresh.
10m
Reduction and alkylation:
Reduction and alkylation:
1h 20m
1h 20m
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.
Reduce your protein sample.
Note
A final concentration (Fc) of 5 millimolar (mM) of DTT is used for reducing proteins.
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.
For 130 µL of lysate, add (5 * 130) / 100, or 6.5 µL of stock 100 millimolar (mM) DTT.
For the 500 µL lysate, add 25 µL of the stock 100 millimolar (mM) DTT.
Place the tubes on the Eppendorf thermomixer and heat for 00:10:00 at 60 °C .
Note
This reduces the cysteine bonds.
10m
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.
For 130 µL of protein sample, add (15 * 130) / 100 or 20 µL of IAM to the samples, and for 500 µL of sample, add (15 * 500) / 100 or 75 µL of IAM to the samples.
Vortex mix the tubes briefly and place the protein sample tubes in the dark (a drawer) for 00:30:00 .
30m
After 00:30:00 , 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.
Note
Do not incubate your protein lysate in the presence of IAM for longer than 30 minutes. Extended incubation may result in unwanted side-chain modifications of amino acids other than cysteines.
30m
Centrifuge your samples at 14000 rcf for 00:10:00 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!
Note
Hereus Megafuge 16R
10m
Remove the supernatant using a clean pipette tip to a labelled tube. This is the protein lysate.
10m
You have now reduced and alkylated your protein lysate samples.
Note
You may now freeze the samples at this stage, or proceed to protein quantitation using the Millipore Direct Detect.