Protocol of 4-plex N,N-Dimethyl Leucine Isobaric Labeling Based Proteome Quantitation by LC-MS/MS

Isobaric labeling is one of the wide-spread high throughput proteome quantification strategies, where enzymatically digested protein extracts from different experimental groups are labeled with chemical tags and yield identical mass shift for individual peptides in MS precursor scan, whereas quantitation can be achieved by comparing reporter ion ratios upon fragmentation in tandem MS scan. In addition to commercial tags such as TMT and iTRAQ, we developed 4-plex and 12-plex N,N-dimethyl leucine (DiLeu) tags as cost-effective alternatives with comparable performance 1,2. DiLeu labeling methods allow multiplexing of samples and enable comparative analysis of acetyl-CoA flux regulated proteomes from different mouse models in the same LC-MS runs, providing statistically reliable data that avoids run-to-run variations. This protocol describes steps including 40 hrs of sample preparation of lysis, tryptic digestion, tag labeling, SCX purification and high pH fractionation, as well as details about LC-MS/MS analysis and data processing. The protocol can be applied to other proteome quantitation systems.