title: Super-SILAC mix coupled with SIM/AIMS assays for targeted verification of phosphopeptides discovered in a large-scale phosphoproteome analysis of hepatocellular carcinoma.
abstract: 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.
  
  
  
  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.
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Yu-Tsun
authors.initials: YT
authors.lastname: Lin
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Clinical Proteomics Core Laboratory, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Kun-Yi
authors.initials: KY
authors.lastname: Chien
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Chia-Chun
authors.initials: CC
authors.lastname: Wu
authors.affiliation: Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Wen-Yu
authors.initials: WY
authors.lastname: Chang
authors.affiliation: Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan.
authors.forename: Lichieh Julie
authors.initials: LJ
authors.lastname: Chu
authors.affiliation: Department of Public Health and Biostatistics Consulting Center, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Min-Chi
authors.initials: MC
authors.lastname: Chen
authors.affiliation: Department of Biochemistry, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan. Electronic address: chautingy@gmail.com.
authors.forename: Chau-Ting
authors.initials: CT
authors.lastname: Yeh
authors.affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan; Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taiwan. Electronic address: yusong@mail.cgu.edu.tw.
authors.forename: Jau-Song
authors.initials: JS
authors.lastname: Yu
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002852:Chromatography, Ion Exchange / Q000379:methods; D005260:Female; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008297:Male; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009363:Neoplasm Proteins; D010750:Phosphoproteins; D020543:Proteome
keywords: Hepatocellular carcinoma; Quantitative phosphoproteomics; SIM/AIMS; Super-SILAC mixture; Targeted verification