An informatics-assisted label-free quantitation strategy that depicts phosphoproteomic profiles in lung cancer cell invasion

Yi Ting Wang, Chia Feng Tsai, Tzu Chan Hong, Chih Chiang Tsou, Pei Yi Lin, Szu Hua Pan, Tse Ming Hong, Pan Chyr Yang, Ting Yi Sung, Wen Lian Hsu, Yu Ju Chen

研究成果: Article

49 引文 斯高帕斯(Scopus)


Aberrant protein phosphorylation plays important roles in cancer-related cell signaling. With the goal of achieving multiplexed, comprehensive, and fully automated relative quantitation of site-specific phosphorylation, we present a simple label-free strategy combining an automated pH/acid-controlled IMAC procedure and informatics-assisted SEMI (sequence, elution time, mass-to-charge, and internal standard) algorithm. The SEMI strategy effectively increased the number of quantifiable peptides more than 4-fold in replicate experiments (from 262 to 1171, p < 0.05, false discovery rate = 0.46%) by using a fragmental regression algorithm for elution time alignment followed by peptide cross-assignment in all LC-MS/MS runs. In addition, the strategy demonstrated good quantitation accuracy (10-12%) for standard phosphoprotein and variation less than 1.9 fold (within 99% confidence range) in proteome scale and reliable linear quantitation correlation (R2 = 0.99) with 4000-fold dynamic concentrations, which was attributed to our reproducible experimental procedure and informatics-assisted peptide alignment tool to minimize system variations. In an attempt to explore metastasis-associated phosphoproteomic alterations in lung cancer, this approach was used to delineate differential phosphoproteomic profiles of a lung cancer metastasis model. Without sample fractionation, the SEMI algorithm enabled quantification of 1796 unique phosphopeptides (false discovery rate = 0.56%) corresponding to 854 phosphoproteins from a series of non-small cell lung cancer lines with varying degrees of in vivo invasiveness. Nearly 40% of the phosphopeptides showed >2-fold change in highly invasive cells; validation of phosphoprotein subsets by Western blotting not only demonstrated the consistency of data obtained by our SEMI strategy but also revealed that such dramatic changes in the phosphoproteome result mostly from translational or post-translational regulation. Mapping of these differentially expressed phosphoproteins in multiple cellular pathways related to cancer invasion and metastasis suggests that the site and degree of phosphorylation might have distinct patterns or functions in the complex process of cancer progression.

頁(從 - 到)5582-5597
期刊Journal of Proteome Research
出版狀態Published - 2010 十一月 5


All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)


Wang, Y. T., Tsai, C. F., Hong, T. C., Tsou, C. C., Lin, P. Y., Pan, S. H., Hong, T. M., Yang, P. C., Sung, T. Y., Hsu, W. L., & Chen, Y. J. (2010). An informatics-assisted label-free quantitation strategy that depicts phosphoproteomic profiles in lung cancer cell invasion. Journal of Proteome Research, 9(11), 5582-5597.