6-Plex Tandem Phosphorus Tags (TPT) for Accurate Quantitative Proteomics.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18 O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H 2 18 O. The combinational incorporations of 18 O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.