Identification and quantification of low abundance growth factors and regulators in complex biological samples still present a challenging task in analytical biochemistry. Immunoassays are often used for such purpose ...Identification and quantification of low abundance growth factors and regulators in complex biological samples still present a challenging task in analytical biochemistry. Immunoassays are often used for such purpose but immunoassays face limitation of both availability and qualities of antibody reagents that are necessary for development of immune assays. With genomics data base available, mass spectrometry (MS) can analyze protein tryptic peptides directly for quantitative determination of proteins. In this study, we report a method for detection of matrix metalloproteinase 1 (MMP1), an important extracellular matrix modulator, in human breast cancer cells by quadrupole time-of-flight (Q-TOF) MS. Absolute quantification of MMP1 was conducted using the selected reaction monitoring (SRM) on a triple quadrupole (Triple-Quad) MS via transitions selected from MMP1 tryptic peptides using non isotope labeled MMP1 protein as a titration standard. In comparison with immune based assay, this MS method showed picogram level sensitivity for quantitative determination of MMP1 intotal cell lysates. Our results demonstrated the feasibility of absolute quantification of low abundance proteins using label-free protein standard by mass spectrometry. Therefore, this method provides not only advantages of high sensitivity but also cost saving in comparison with the commonly used mass spectrometry that currently employs isotype labeled proteins for quantitative analysis.展开更多
目的：利用经典核雌激素受体（ER）阴性、膜G蛋白偶联雌激素受体（GPER）阳性乳腺癌SKBR3细胞探索丹参酮ⅡA（TanshinoneⅡA）对肿瘤细胞增殖活性的影响及其GPER介导功能。方法：应用GPER Si RNA转染构建GPER基因沉默的SKBR3细胞,并用We...目的：利用经典核雌激素受体（ER）阴性、膜G蛋白偶联雌激素受体（GPER）阳性乳腺癌SKBR3细胞探索丹参酮ⅡA（TanshinoneⅡA）对肿瘤细胞增殖活性的影响及其GPER介导功能。方法：应用GPER Si RNA转染构建GPER基因沉默的SKBR3细胞,并用Western Blot方法检测基因沉默效果;利用MTT细胞增殖速率、PI细胞周期测定方法观察丹参酮ⅡA对SKBR3细胞增殖速率、细胞增殖指数的影响及GPER的介导作用。利用Western Blot方法检测丹参酮ⅡA对SKBR3细胞周期蛋白Cyclin D表达的影响及GPER的介导功能。结果：（1×10^-5～1×10^-8）mol/L丹参酮ⅡA能够显著降低SKBR3细胞增殖速率,（1×10^-5～1×10^-6）mol/L丹参酮ⅡA能够显著下调细胞增殖指数,且上述抑制作用可因GPER基因沉默而明显减弱。Western Blot测定结果表明,（1×10^-5～1×10^-6）mol/L丹参酮ⅡA可使SKBR3细胞Cyclin D蛋白表达显著降低,且该效应可被GPER基因沉默所拮抗。结论：丹参酮ⅡA具有抑制乳腺癌SKBR3细胞增殖的作用,该抑制作用是经GPER途径介导的。展开更多
文摘Identification and quantification of low abundance growth factors and regulators in complex biological samples still present a challenging task in analytical biochemistry. Immunoassays are often used for such purpose but immunoassays face limitation of both availability and qualities of antibody reagents that are necessary for development of immune assays. With genomics data base available, mass spectrometry (MS) can analyze protein tryptic peptides directly for quantitative determination of proteins. In this study, we report a method for detection of matrix metalloproteinase 1 (MMP1), an important extracellular matrix modulator, in human breast cancer cells by quadrupole time-of-flight (Q-TOF) MS. Absolute quantification of MMP1 was conducted using the selected reaction monitoring (SRM) on a triple quadrupole (Triple-Quad) MS via transitions selected from MMP1 tryptic peptides using non isotope labeled MMP1 protein as a titration standard. In comparison with immune based assay, this MS method showed picogram level sensitivity for quantitative determination of MMP1 intotal cell lysates. Our results demonstrated the feasibility of absolute quantification of low abundance proteins using label-free protein standard by mass spectrometry. Therefore, this method provides not only advantages of high sensitivity but also cost saving in comparison with the commonly used mass spectrometry that currently employs isotype labeled proteins for quantitative analysis.
