Proteomics

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Reproducibility of label-free single-shot phosphoproteomics applied to CRC cell lines

ABSTRACT: There is a need for robust phosphopeptide enrichment methods to allow signaling network analysis in cancer cell lines and tissues with minimal fractionation. With recent instrument developments thousands of unique phosphopeptides can be detected by single-shot LC-MS/MS. However, successful phosphoproteomics experiments still rely on efficient phosphopeptide enrichment from a tryptic digest prior to LC-MS/MS analysis. Here we describe a performance assessment of HAMMOC (hydroxyl acid modified metal affinity chromatography) (Sugiyama MCP2007, Kyono, JPR 2008) combined with single shot label-free quantitation at 500 µg peptide input level. In a triplicate analysis we observe good phosphopeptide identification reproducibility (75.8%), depth of identification (6014-6150 phosphopeptides) and reproducibility of label-free quantification (CV 17.8%, Pearson r 0.87-0.98) by single-shot LC-MS/MS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Connie Ramona Jimenez  

PROVIDER: MSV000080715 | MassIVE | Tue Mar 28 23:46:00 BST 2017

SECONDARY ACCESSION(S): PXD001546

REPOSITORIES: MassIVE

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Publications

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

Piersma Sander R SR   Knol Jaco C JC   de Reus Inge I   Labots Mariette M   Sampadi Bharath K BK   Pham Thang V TV   Ishihama Yasushi Y   Verheul Henk M W HM   Jimenez Connie R CR  

Journal of proteomics 20150401 Pt B

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 glob  ...[more]