Super-SILAC mix coupled with SIM/AIMS assays for targeted verification of phosphopeptides discovered in a large-scale phosphoproteome analysis of hepatocellular carcinoma

J Proteomics. 2017 Mar 22;157:40-51. doi: 10.1016/j.jprot.2017.02.005. Epub 2017 Feb 10.


Plentiful studies have established a close association between aberrant phosphorylation and hepatocellular carcinoma (HCC). Here, we applied a quantitative phosphoproteomics platform combining dimethylation labeling and online 3D strong cation exchange chromatography (SCX)-titanium oxide (TiO2)/RP-LTQ-Orbitrap to compare phosphoproteomes between three pairs of HCC tissues and non-tumor counterparts. This analysis yielded 7868 quantifiable phosphopeptides and numerous up- or down-regulated candidates. Increased phosphorylation of LMNA and NIPA was confirmed using specific antibodies. To expand our verification capability, we evaluated the use of LTQ-Orbitrap run in SIM/Accurate inclusion mass screening (AIMS) mode with a super-SILAC mixture as an internal standard to quantify a subset of phosphopeptide candidates in HCC tissue samples. In sample I used for discovery experiment, we successfully quantified 32 (in SIM mode) and 30 (in AIMS mode) phosphopeptides with median coefficients of variation (CVs) of 7.5% and 8.3%, respectively. When the assay was applied to other three pairs of HCC specimens for verification experiment, 40 target phosphopeptides were quantified reliably (~7.5% CV), and more than half of them were differentially expressed between tumor and adjacent non-tumor tissues. Collectively, these results indicate the feasibility of using super-SILAC mix-SIM/AIMS assays for targeted verification of phosphopeptides discovered by large-scale phosphoproteome analyses of HCC specimens.

Significance: In this study, we developed a strategy for conducting both discovery and targeted verification of deregulated phosphoproteins in HCC tissue specimens on LTQ-Orbitrap. This strategy allowed us to generate a quantitative HCC tissue phosphoproteome dataset containing significantly deregulated phosphoproteins that represents a valuable resource for the identification of potential HCC biomarkers and/or therapeutic targets. Furthermore, our proof-of-concept experiments demonstrated the feasibility of applying LTQ-Orbitrap, operated in SIM/AIMS mode, to multiplex and targeted verification of phosphopeptides in individual tissue specimens using a super-SILAC mix as an internal phosphopeptide standard. This method could be readily applied to verify dozens of phosphopeptide candidates in a larger HCC sample set.

Keywords: Hepatocellular carcinoma; Quantitative phosphoproteomics; SIM/AIMS; Super-SILAC mixture; Targeted verification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Hepatocellular / metabolism*
  • Carcinoma, Hepatocellular / pathology
  • Chromatography, Ion Exchange / methods
  • Female
  • Humans
  • Liver Neoplasms / metabolism*
  • Liver Neoplasms / pathology
  • Male
  • Neoplasm Proteins / isolation & purification
  • Neoplasm Proteins / metabolism*
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proteome / isolation & purification
  • Proteome / metabolism*


  • Neoplasm Proteins
  • Phosphoproteins
  • Proteome