Assessing Budding Yeast Phosphoproteome Dynamics in a Time-Resolved Manner using TMT10plex Mass Tag Labeling

Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS). We have used this technology to label 20 Saccharomyces cerevisiae samples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological and mass spectrometry sample preparation and analysis. For complete details on the use and execution of this protocol, please refer to Touati et al. (2019). ▪ •Characterization of the phosphoproteome in a time-resolved manner•TMT10plex Mass Tag labelling of budding yeast extracts•Mass spectrometry sample preparation and analysis Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS).We have used this technology to label 20 Saccharomyces cerevisiae samples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological andmass spectrometry sample preparation and analysis.