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

Yu Tsun Lin, Kun Yi Chien, Chia Chun Wu, Wen Yu Chang, Lichieh Julie Chu, Min Chi Chen, Chau Ting Yeh*, Jau Song Yu

*此作品的通信作者

研究成果: 期刊稿件文章同行評審

7 引文 斯高帕斯(Scopus)

摘要

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.

原文英語
頁(從 - 到)40-51
頁數12
期刊Journal of Proteomics
157
DOIs
出版狀態已出版 - 22 03 2017

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Publisher Copyright:
© 2017 Elsevier B.V.

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