The Use of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Study Ivermectin-Mediated Molecular Pathway Changes in Human Ovarian Cancer Cells

Na Li,Xianquan Zhan

doi:10.5772/intechopen.102092

Abstract

Stable isotope labeling with amino acids in cell culture (SILAC) was to use isotopic essential amino acids to replace the original amino acids for cell culture and passage for 8–10 generations, followed by mass spectrometry to identify proteins and the isotopic abundance difference to quantify proteins. SILAC can be used to characterize proteomic changes, and analyze protein turnover, protein interactions, and dynamic changes with quantitative accuracy, and high reproducibility. For this study, SILAC “light” (L-Lysine-2HCl [12C6, 14N2], L-Arginine-HCl [12C6, 14N4])- or “heavy” (L-Lysine-2HCl [13C6, 15N2], L-Arginine-HCl [13C6, 15N4])-labeling RPMI 1640 medium was used to culture human ovarian cancer TOV-21G cells for 10 passages, followed by the treatment of 0.1% dimethylsulfoxide for 24 h and 20 µM ivermectin for 24 h, respectively. The light- and heavy-isotope-labeled proteins were equally mixed (1:1) for digestion with trypsin. The tryptic peptide mixture was fractionated with liquid chromatography and analyzed with tandem mass spectrometry. In total, 4,447 proteins were identified in ivermectin-treated TOV-21G cells in relation to controls. Those proteins were enriched in 89 statistically significant signaling pathways and 62 statistically significant biological processes. These findings clearly demonstrated that SILAC quantitative proteomics was a useful and reliable method to study ivermectin-related proteomic changes in cancer cells, which in combination with molecular pathway networks and biological processes enrichments provided more comprehensive insights into molecular mechanisms of ivermectin in inhibiting TOV-21G cells.

Highlights

Stable isotope labeling with amino acids in cell culture (SILAC) is a polypeptidelabeling technology developed by the Thermo Fisher company of the United States in 2002 [1]
The human ovarian cell line (TOV-21G) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 5% CO2 at 37°C. (i) Ovarian cell line used here was TOV-21G, which was obtained from Keibai Academy of Science (Nanjing, China) [18]. (ii) RPMI 1640 was used without glutamine, lysine, and arginine. (iii) FBS was brought from Gibco® Certified Thermo Fisher Scientific. (iv) The growing states of TOV-21G were observed, and the medium was changed in every 2 days
4447 proteins were identified with SILAC quantitative proteomics in human ovarian cancer cells treated with ivermectin

Summary

Introduction

Stable isotope labeling with amino acids in cell culture (SILAC) is a polypeptidelabeling technology developed by the Thermo Fisher company of the United States in 2002 [1]. Heavy isotopes (13C or 15N) and light isotopes (12C or 14N) are used to label two essential amino acids (L-lysine and L-arginine) that are contained in a cell-cultured medium, respectively. After the cells were cultured with essential amino acids for 6–10 generations, all proteins were labeled with heavy isotopes or light isotopes. SILAC was widely used in quantitative proteomics to study pathogenesis, drug target, protein modification and dynamics, protein-molecule interaction, and screen special functional proteins [4]. SILAC showed outstanding performance for quantification and dynamics of phosphosites in colorectal cancer with the treatment of the epidermal growth factor receptor (EGFR)-blocking antibody cetuximab, rendering it the effective method for cellular signaling study in cell culture models [5]. An MS-based approach combining dynamic-SILAC labeling with isobaric mass tagging was well used to understand protein degradation and synthesis in cellular systems [10]. SILAC provided an effective scheme to comprehensively and systematically qualify and quantify complex mammalian cell proteome, which would promote progress in the medical field

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