A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification
A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification
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DOI：
10.1002/pmic.200300606
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发表时间：
2004-03-01
期刊：
影响因子：
3.4
通讯作者：
Lubman, DM
中科院分区：
文献类型：
--
作者：
Hamler, RL;Zhu, K;Lubman, DM
提出了一种用于分析正常和恶性乳腺上皮细胞系细胞裂解产物的二维液相分离与质谱仪联用的方法。液相分离包括第一维等电聚焦和第二维无孔硅胶反相高效液相色谱(NPS-RP-HPLC)。用电喷雾电离飞行时间质谱仪(ESI-TOF MS)进行蛋白质定量和质量检测。采用基质辅助激光解吸电离飞行时间MS(MALDI-TOF MS)和MALDI-四极飞行时间(QTOF)-串联质谱仪(MS/MS)鉴定蛋白质。选择了两个pH区，每个pH区有50-60个独特的蛋白质用于分析。创建了质谱图，使正常和恶性乳腺上皮细胞之间的蛋白质定量差异得以可视化。在有限的pH范围内的质量图谱实验中观察到的大约110个独特的蛋白质中，40个(36%)通过肽质量指纹图谱得到了肯定的鉴定，并被归类到质谱图上的区带。在这40种蛋白质中，有22种在一种或多种恶性细胞系中高表达。这些蛋白质是潜在的乳腺癌生物标志物，可能有助于诊断、治疗或药物开发。
A two-dimensional liquid-phase separation scheme coupled with mass spectrometry (MS) is presented for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid-phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) as the second dimension. Protein quantitation and mass,, measurement are performed using electrospray ionization-time of flight MS (ESI-TOF MS). Proteins are identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS) and MALDI-quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS). Two pH regions with 50-60 unique proteins in each pH range were chosen for analysis. Mass maps were created that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development.