Aug 09, 2023
TMT labelling
- 1Lazarou Lab, WEHI
Protocol Citation: Louise Uoselis 2023. TMT labelling. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmp69l5b/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: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86211
Keywords: ASAPCRN, labelling tmt, tmt
Abstract
TMT labelling
Reconstitute lyophilised peptides in 25 µL of 100 mM TEAB by vortexing each sample for ~00:00:10 , and then leaving to sit at Room temperature for 00:15:00 . Each sample was then sonicated for 00:05:00 in a waterbath sonicator in an ice slurry.
20m 10s
Determine the peptide concentration of each sample spectroscopically
Aliquot10 µg of peptides for each sample into a LoBind microfuge tube and add 100 millimolar (mM) triethylammonium bicarbonate to a final volume of 100 µL for each sample.
For the pooled batch control, divide 100 µg by the total number of samples, and aliquot that amount of peptides from each sample into one tube. Make up the pooled control volume to 100 µL with 100 millimolar (mM) triethylammonium bicarbonate (can lyophilise the samples to reduce the volume if the total pooled volume exceeds 100 uL).
Bring the TMT labels to Room temperature .
Reconstitute each label in acetonitrile as per the manufacturer’s instructions.
Add the required volume of the designated label to each sample, and vortex each sample for ~ 00:00:05 to mix.
5s
Leave samples to incubate at Room temperature for01:00:00 (static).
1h
Add hydroxylamine to a final concentration of 0.26%/sample, vortex each sample for ~00:00:05 , and incubate at Room temperature for00:15:00 to quench the TMT labelling reaction.
15m 5s
Combine the samples into their designated batches, and lyophilise the pooled samples. Seal each tube with parafilm and store at -80 °C for downstream processing.