Toward a Comprehensive Characterization of a Human Cancer Cell Phosphoproteome

Mass spectrometry (MS)-based phosphoproteomics has achieved
extraordinary
success in qualitative and quantitative analysis of cellular protein
phosphorylation. Considering that an estimated level of phosphorylation
in a cell is placed at well above 100 000 sites, there is still
much room for improvement. Here, we attempt to extend the depth of
phosphoproteome coverage while maintaining realistic aspirations in
terms of available material, robustness, and instrument running time.
We developed three strategies, where each provided a different balance
between these three key parameters. The first strategy simply used
enrichment by Ti4+-IMAC followed by reversed chromatography
LC–MS (termed 1D). The second strategy incorporated an additional
fractionation step through the use of HILIC (2D). Finally, a third
strategy was designed employing first an SCX fractionation, followed
by Ti4+-IMAC enrichment and additional fractionation by
HILIC (3D). A preliminary evaluation was performed on the HeLa cell
line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy
was found to be the most sensitive but limited in comprehensivity,
mainly due to issues with complexity and dynamic range. Overall, the
best balance was achieved using the 2D based strategy, identifying
close to 17 000 phosphopeptides with less than 1 mg of material
in about 48 h. Subsequently, we confirmed the findings with the K562
cell sample. When sufficient material was available, the 3D strategy
increased phosphoproteome allowing over 22 000 unique phosphopeptides
to be identified. Unfortunately, the 3D strategy required more time
and over 1 mg of material before it started to outperform 2D. Ultimately,
combining all strategies, we were able to identify over 16 000
and nearly 24 000 unique phosphorylation sites from the cancer
cell lines HeLa and K562, respectively. In summary, we demonstrate
the need to carry out extensive fractionation for deep mining of the
phosphoproteome and provide a guide for appropriate strategies depending
on sample amount and/or analysis time.