Well, look no further because we’ve got a solution for ya: multiplexed protein quantitation using amine-reactive isobaric tagging reagents in Saccharomyces cerevisiae!
Before anything else, what these fancy words mean. Isobaric tags are basically chemical labels that allow you to identify and measure multiple proteins simultaneously because they have the same mass but different chemical properties (hence “isobaric”). And amine-reactive means that they react with primary amines, which are found on the surface of most proteins.
So how does this work in yeast? Well, we’ve got a protocol for you! Here’s what you need:
1️ Grow your yeast cells to log phase (around 2-3 million cells/mL) and harvest them by centrifugation.
2️ Resuspend the pellet in lysis buffer containing urea, Triton X-100, and protease inhibitors. Add your isobaric tagging reagents (e.g., iTRAQ or TMT) to each sample according to manufacturer’s instructions.
3️ Incubate the samples at room temperature for 1 hour with gentle shaking, then quench any remaining reactive groups by adding a solution of hydroxylamine (e.g., 50 mM in water) and incubating for another 15 minutes.
4️ Perform SDS-PAGE or gel electrophoresis to separate the proteins based on their molecular weight, then excise the desired bands/regions using a clean scalpel blade.
5️ In-gel digestion: add trypsin (or another suitable enzyme) and incubate overnight at 37°C to cleave the proteins into smaller peptides.
6️ Extract the resulting peptide mixture using a solution of acetonitrile/water, then dry it down in a vacuum centrifuge or speedvac.
7️ Reconstitute the dried peptides in buffer (e.g., water with formic acid) and analyze them by mass spectrometry using a triple quadrupole or other suitable instrument.
8️ Data analysis: use software tools like MaxQuant, Proteome Discoverer, or Skyline to identify and quantify the peptides based on their isobaric tagging labels (e.g., iTRAQ or TMT).
And that’s it! You now have multiplexed protein quantitation data for your yeast samples using amine-reactive isobaric tagging reagents.