Solid Tumor Proteome and Phosphoproteome Analysis by High Resolution Mass Spectrometry

Kinases play a prominent role in tumor development, pointing to the presence of specific phosphorylation patterns in tumor tissues. Here, we investigate whether recently developed high resolution mass spectrometric (MS) methods for proteome and phosphoproteome analysis can also be applied to solid tumors. As tumor model, we used TG3 mutant mice carrying skin melanomas. At total of 100 μg of solid tumor lysate yielded a melanoma proteome of 4443 identified proteins, including at least 88 putative melanoma markers previously found by cDNA microarray technology. Analysis of 2 mg of lysate from dissected melanoma with titansphere chromatography and 8 mg with strong cation exchange together resulted in the identification of more than 5600 phosphorylation sites on 2250 proteins. The phosphoproteome included many hits from pathways important in melanoma. One-month storage at −80 °C did not significantly decrease the number of identified phosphorylation sites. Thus, solid tumor can be analyzed by MS-based proteomics with similar efficiency as cell culture models and in amounts compatible with biopsies.

激酶在肿瘤发生发展中发挥关键作用，这提示肿瘤组织中存在特异性的磷酸化修饰模式。本研究旨在探讨近年来开发的用于蛋白质组与磷酸化蛋白质组分析的高分辨率质谱（MS）技术，是否可应用于实体肿瘤的相关分析。本研究以携带皮肤黑色素瘤的TG3突变小鼠作为肿瘤模型。通过对总计100 μg实体肿瘤裂解液进行分析，共鉴定出4443个黑色素瘤蛋白质组相关蛋白，其中包含至少88个此前通过cDNA微阵列技术发现的潜在黑色素瘤标志物。采用泰坦球色谱（Titansphere Chromatography）对2 mg解剖分离的黑色素瘤裂解液进行分析，结合强阳离子交换色谱（strong cation exchange）对8 mg裂解液进行分析，最终共鉴定出2250个蛋白上的5600余个磷酸化位点。该磷酸化蛋白质组涵盖了诸多与黑色素瘤发生发展密切相关的信号通路相关靶点。样品在−80 ℃条件下储存1个月，并未显著降低可鉴定的磷酸化位点数量。综上，基于质谱的蛋白质组学技术可用于实体肿瘤分析，其鉴定效率与细胞培养模型相当，且所需样品量与活检样本兼容。