title: Application of the SILAC (stable isotope labelling with amino acids in cell culture) technique in quantitative comparisons for tissue proteome expression.
abstract: Stable isotope labelling has recently become a popular tool for the quantitative profiling of the proteome, especially the emergence and development of the SILAC (stable isotope labelling with amino acids in cell culture) technique. Here we have expanded the application of SILAC to comparison of the relative protein expression levels between two different states of tissues based on cultured cells with [2H]leucine labelling as an internal standard in mass spectra. The SILAC ratio of tissue proteins versus labelled cells was determined by the calculation of peak intensity of the pair of labelled and unlabelled peptide fragment ions from the mass spectra, and the relative expression level of proteins in two groups of tissues was estimated by calculating the ratio of their SILAC ratio. To validate our [2H]leucine-based differential proteome analysis for tissues, we successfully compared two known proteins, one up-regulated vimentin and one down-regulated enoyl-CoA hydratase in human renal cancerous tissues versus human normal kidney tissues, which was previously confirmed by other groups using conventional two-dimensional PAGE analysis. Furthermore, we identified a previously unknown down-regulated protein, COX4I1 (cytochrome c oxidase subunit 4 isoform 1), in renal carcinoma tissues by this [2H]leucine-based quantitative proteomics method, which was also validated by immunohistochemistry and Western-blot analysis. In conclusion, the application of the [2H]leucine-based quantitative technique can be effectively expanded to comparison of the expression levels for the tissue proteome at different states, which would help us to identify new candidate biomarkers for tumours.
authors.affiliation: West China Hospital, Sichuan University, Chengdu, People's Republic of China.
authors.forename: Yuhuan
authors.initials: Y
authors.lastname: Xu
authors.forename: Shufang
authors.initials: S
authors.lastname: Liang
authors.forename: Guobo
authors.initials: G
authors.lastname: Shen
authors.forename: Xuejiao
authors.initials: X
authors.lastname: Xu
authors.forename: Qingping
authors.initials: Q
authors.lastname: Liu
authors.forename: Zhizhong
authors.initials: Z
authors.lastname: Xu
authors.forename: Fengming
authors.initials: F
authors.lastname: Gong
authors.forename: Minghai
authors.initials: M
authors.lastname: Tang
authors.forename: Yuquan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D003903:Deuterium / Q000378:metabolism; D015536:Down-Regulation; D003576:Electron Transport Complex IV / Q000032:analysis / Q000378:metabolism; D004746:Enoyl-CoA Hydratase / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007553:Isotope Labeling / Q000379:methods*; D007680:Kidney Neoplasms / Q000737:chemistry / Q000378:metabolism; D007930:Leucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis* / Q000096:biosynthesis; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000032:analysis / Q000378:metabolism
chemical_list: D020543:Proteome; D014746:Vimentin; D003903:Deuterium; D003576:Electron Transport Complex IV; D004746:Enoyl-CoA Hydratase; D007930:Leucine