Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome

Protein phosphorylation is a prevalent post-translational
modification
that regulates essentially every aspect of cellular processes. Currently,
liquid
chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive
offline sample fractionation and a phosphopeptide enrichment method
is a best practice for deep phosphoproteome profiling, but balancing
throughput and profiling depth remains a practical challenge. We present
an online three-dimensional separation method for ultradeep phosphoproteome
profiling that combines an online two-dimensional liquid chromatography
separation and an additional gas-phase separation. This method identified
over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells
during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome
significantly expanded the detectable functional landscape of cellular
phosphoproteome.