Aug 09, 2023

Public workspaceTMT labelling

DOI
dx.doi.org/10.17504/protocols.io.n92ldmp69l5b/v1
  • 1Lazarou Lab, WEHI
Louise Uoselis
Open access
DOI: dx.doi.org/10.17504/protocols.io.n92ldmp69l5b/v1
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
Abstract
TMT labelling
Reconstitute lyophilised peptides in Amount25 µL of 100 mM TEAB by vortexing each sample for ~Duration00:00:10 , and then leaving to sit at TemperatureRoom temperature for Duration00:15:00 . Each sample was then sonicated for Duration00:05:00 in a waterbath sonicator in an ice slurry.

20m 10s
Determine the peptide concentration of each sample spectroscopically
AliquotAmount10 µg of peptides for each sample into a LoBind microfuge tube and add Concentration100 millimolar (mM) triethylammonium bicarbonate to a final volume of Amount100 µL for each sample.

For the pooled batch control, divide Amount100 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 Amount100 µL with Concentration100 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 TemperatureRoom 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 ~ Duration00:00:05 to mix.

5s
Leave samples to incubate at TemperatureRoom temperature forDuration01:00:00 (static).

1h
Add hydroxylamine to a final concentration of 0.26%/sample, vortex each sample for ~Duration00:00:05 , and incubate at TemperatureRoom temperature forDuration00:15:00 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 Temperature-80 °C for downstream processing.