This familiarization tutorial provides a step-by-step instructions for using the Proteome Discoverer application's reporter ion quantification capabilities to analyze a dataset containing multiple plexes combined into a single experiment.

TIP

New users might want to complete a single plex exercise before doing a multiplex exercise. For a single plex TMT exercise, see Analysis of yeast gene knockouts by multiplexed reporter ion quantification.

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 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, TKOTT11_1ms3_2.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 LC/MS run

Disk space:

2 GB local drive space, SSD drive preferred

Processing time:

Approximately 10 minutes