The most accurate quantitation on protein complex mixtures can be accomplished by employing TMT SPS MS3 method on Tribrid mass spectrometers. However, this method is not optimal for phosphopeptide quantitation due to special characteristics in phosphopeptide fragmentation patterns. In this study, we developed methods to address this limitation and provide high phosphopeptide identifications and accurate quantitation.
Digested HeLa cells were labeled with TMT10plex™ reagents and mixed at ratios of 16:8:4:2:1:1:2:4:8:16. Yeast digest was labeled with the last 5 channels mixed equimolar (0:0:0:0:0:1:1:1:1:1) and spiked into TMT-labeled HeLa digest sample as interference. This resulted in Hela digests with first five channels that were free of interference and the last five channels interfered by yeast proteome. The mixed samples were further enriched for phosphopeptides and analyzed on Orbitrap Fusion and Lumos MS.
Ratio distortion was observed for phosphopeptide analysis on HeLa and yeast mixture, when using MS2 workflow. This was due to interfering ions co-isolated with precursor ions. The use of SPS MS3 improved quantitation accuracy. However there was 50% of loss in the number of phosphopeptides identified. The loss was due to the strong presence of the neutral loss peak specific to phosphopeptide, which limited the identifications from CID MS2 spectra. We thus developed and optimized two new MS3 instrument methods to reduce the loss of phosphopeptide identifications. This approach minimized loss to less than 30%, while maintaining quantitation accuracy benefits given by SPS MS3.
The new MS3 methods were applied to large scale phosphoproteome characterization in A549 cell line upon insulin and IGF-1 treatments. Overall, 3,378 protein groups and 12,465 phosphopeptides were identified of which 10,436 were quantifiable. The accurate and reproducible measurement enabled mapping regulated phosphorylation sites to numerous signaling pathways including mTOR signaling and AMPK signaling pathways.