A six-plex proteome quantification strategy reveals the dynamics of protein turnover

MS1 full scan based quantification is one of the most popular approaches for large-scale proteome quantification. Typically only three different samples can be differentially labeled and quantified in a single experiment. Here we present a two stages stable isotope labeling strategy which allows six different protein samples (six-plex) to be reliably labeled and simultaneously quantified at MS1 level. Briefly in the first stage, isotope lysine-d0 (K0) and lysine-d4 (K4) are in vivo incorporated into different protein samples during cell culture. Then in the second stage, three of K0 and K4 labeled protein samples are digested by lysine C and in vitro labeled with light (2CH(3)), medium (2CD(2)H), and heavy (2(13)CD(3)) dimethyl groups, respectively. We demonstrated that this six-plex isotope labeling strategy could successfully investigate the dynamics of protein turnover in a high throughput manner.