This familiarization tutorial provides step-by-step instructions for using the Proteome Discoverer application's reporter ion quantification capabilities to analyze three different individual yeast gene knockouts together with controls in a single multiplex LCMS experiment.

This familiarization exercise uses TMT 11plex quantitation. The sample used in this exercise was acquired using Thermo Scientific Orbitrap Fusion™ Lumos™ mass spectrometer.

For general information about TMT-based quantitation, go to https://www.analyteguru.com/.

For information about purchasing the Pierce™ triple knockout yeast standard, go to https://www.thermofisher.com/order/catalog/product/A40938.

Summary of example analysis

Quan method:

TMT 11plex

Quan channels:

126, 127N, 127C =ME---T6

128N, 128C, 129N = HIS4

129C, 130N, 130C = URA2

131N, 131C = parental

Organism:

Yeast

Instrument:

Orbitrap Fusion Lumos

Acquisition method:

TMT SPS MS3 (CID MS2, HCD MS3)

RAW file:

TKOTT11_1ms3_1.raw

Located in the application media in the folder: Example RAW Files. Copy to a local directory.

Sample description:

Three yeast culture replicates each of MET6, HIS4, and URA2 single gene knockout strains as well as two culture replicates of the parental strain using TMT11 chemistry for a single LCMS run

Disk space:

1GB local drive space, SSD drive preferred

Processing time:

Approximately 10 minutes