title: An investigation of heat shock protein 27 and P-glycoprotein mediated multi-drug resistance in breast cancer using liquid chromatography-tandem mass spectrometry-based targeted proteomics.
abstract: One missing puzzle piece to study heat shock protein 27 (HSP27) in P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) was the amount of HSP27 and the extent of its phosphorylation in the biological context. Liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based targeted proteomics allows researchers to monitor associated proteins and their modification simultaneously and quantitatively. In this study, a targeted proteomics assay was first developed and validated for the quantification of HSP27 and its phosphorylated forms. Using this assay, the level of HSP27 was determined in non-tumoral cells MCF-10A, parental drug-sensitive cancer cells MCF-7/WT and drug-resistant cancer cells MCF-7/ADR. A decrease of HSP27 expression was observed in P-gp overexpressed MCF-7/ADR cells. A quantitative time-course analysis of both HSP27 and P-gp in doxorubicin (DOX)-treated MCF-7/WT cells also implied that HSP27 may participate in the P-gp modulation. Furthermore, stoichiometry of site-specific HSP27 phosphorylation indicated that DOX treatment rapidly induced the HSP27 phosphorylation at Ser82. Moreover, conventional analytical methods were also performed for a comparison.
  
  
  CONCLUSIONS
  LC/MS/MS-based targeted proteomics turns out to be a promising quantification approach for the study of proteins in the preclinical and clinical environment. Unfortunately, rare studies applied this technology to detect multiple associated proteins or protein modification in one experiment. This study demonstrated the potential of LC/MS/MS-based targeted proteomics to understand the cell events in a more accurate context of biological system. By the quantitative time-course analysis of HSP27 and its phosphorylated forms at sites of Ser15 and Ser82, the possible role of HSP27 in P-gp mediated MDR was suggested. Further development of targeted proteomics in future may provide more insight into signal transduction pathways upon perturbation of a protein network or changes to a panel of proposed biomarkers in a given disease state.
authors.affiliation: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Xu
authors.affiliation: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Yang
authors.affiliation: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, China.
authors.forename: Danjun
authors.initials: D
authors.lastname: Fang
authors.affiliation: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, China.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Xu
authors.affiliation: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing 211166, China. Electronic address: ychen@njmu.edu.cn.
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004317:Doxorubicin / Q000493:pharmacokinetics / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005260:Female; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000903:Antibiotics, Antineoplastic; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D004317:Doxorubicin
keywords: Heat shock protein 27 (HSP27); Liquid chromatography–tandem mass spectrometry; Multi-drug resistance; P-glycoprotein (P-gp); Phosphorylation; Targeted proteomics