Feasibility of label-free phosphoproteomics and application to base-line signaling of colorectal cancer cell lines

• Performance assessment of single-shot LC-MS/MS label-free phosphoproteomics using TiOx-based phosphopeptide enrichment.
• Label-free single-shot phosphoproteomics is a reproducible workflow for quantitative profiling of biological cell lines.
• Label-free phosphopeptide profiling of a panel of 8 colorectal cancer cell lines representing 3 CRC subtypes.
• Phosphopeptide profiles correlate with transcriptome-derived CRC subtypes and confirm EMT properties of the CCS3 subtype.
• Phosphopeptide profiles reveal RNA splicing as one of the discriminating processes.

Robust phosphopeptide enrichment methods with minimal fractionation are required to profile signaling network analysis in cancer cell lines and tissues. We assessed performance of single-shot LC-MS/MS label-free phosphoproteomics using TiOx-based phosphopeptide enrichment and report phosphopeptide identification reproducibility (75.8%), depth of identification (6014–6150 phosphopeptides) and reproducibility of label-free quantification (CV 17.8%). Subsequently, we have profiled the baseline global phosphorylation of 8 colorectal cancer (CRC) cell lines representing different CRC prognostic subtypes. Global single-shot phosphoproteomics can distinguish CRC subtypes previously identified by transcriptomics and identifies signaling proteins and processes associated with the CCS3 poor prognosis subtype. Data are available via ProteomeXchange with identifiers PXD001546 and PXD001550.Biological significanceLabel-free single-shot phosphoproteomics is a mature workflow that can be used for global quantitative profiling of biological cell lines and tissues to map signaling networks in comparative analyses. Here we show the feasibility of label-free profiling of CRC cell lines at sample input levels compatible with clinical samples such as tumor biopsies. This article is part of a Special Issue entitled: HUPO 2014.

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