Phosphorylation is one of the most important post-translational modifications in proteins. It plays a key role in numerous cellular processes, including signal transduction, cell proliferation, and intercellular communication. More than 30% of the cellular proteins are phosphorylated at a given time. However, dysregulation of phosphorylated proteins usually leads to a disorder in the intracellular signaling pathways and the onset of various diseases, especially cancer. Cell proliferation and metastasis are the major manifestations of cancer progression, and these might be affected by the protein phosphorylation levels. Clinically, cancer usually metastasizes at the middle and late stages, affecting other organs beyond primary lesion. This poses significant challenges in cancer treatment and prognosis. Consequently, comparing the phosphorylated proteomes of cells with different metastatic capabilities is helpful in studying the role of protein phosphorylation in cancer metastasis and progression. The human low metastatic lung cancer cell line 95C and high metastatic lung cancer cell line 95D are two of the four sublines isolated from human lung giant cell carcinoma cell line (PLA-801) by the single-cell cloning technique. These are ideal models for studying tumor metastasis and non-small cell lung cancer. MRC-5 cell line was obtained from a 14 week old fetal normal lung tissue. Quantitative analysis of the proteome and phosphorylated proteome in these normal lung cells and lung cancer cells with different metastatic capacities can identify key pathways and regulatory proteins associated with lung cancer metastasis and progression. Immobilized metal affinity chromatography (IMAC) is an efficient technique for the enrichment of phosphopeptides and has been widely used for phosphoproteome research. Metal ions (such as Ti4+) are immobilized on the substrate by chelation, and phosphopeptides can be selectively adsorbed under acidic conditions and eluted under alkaline conditions. IMAC can enrich phosphate groups at different amino acid sites with high specificity. In this study, Ti4+was chelated onto Ti4+-IMAC material, which was used to enrich phosphopeptides for phosphoproteome research. Two enrichment methods, namely, the vortexing method and solid phase extraction (SPE) method, were first compared for the enrichment of phosphopeptides using 10 μm Ti4+-IMAC. Phosphopeptides were highly enriched using the vortexing method. Following this, two sizes of Ti4+-IMAC material (10 μm and 30 μm) were compared to determine the efficiency of phosphopeptide enrichment. Enrichment efficiency was superior with the smaller-sized material. Therefore, the small-size Ti4+-IMAC material was selected for the proteomics research of lung cell phosphorylation. The optimized strategy was further used to compare the phosphoproteomes of the lung cancer cells with different metastatic abilities. Label-free quantification proteomics demonstrated that 510, 863, and 1108 phosphorylated proteins were identified from normal lung fibroblasts (MRC-5), low metastatic lung cancer cells (95C), and high metastatic lung cancer cells (95D), respectively, using the optimized Ti4+-IMAC method. Among them, 317 phosphorylated proteins were shared among the three groups. The protein phosphorylation level increased significantly with increasing cellular metastatic capacity. In our study, 7560 phosphorylation sites were identified on 1268 phosphorylated proteins, among which 1130 phosphorylation sites were differentially expressed. Some abnormally expressed kinases and their phosphorylation levels are closely associated with malignant cell proliferation. Comparative bioinformatics analysis showed that dysregulated phosphoproteins were mainly related to cell migration functions, such as cell invasion, migration, and death. These abnormally expressed phosphorylated proteins and phosphorylation sites could be further validated and studied for lung cancer metastasis. Our study demonstrates that Ti4+-IMAC is a powerful tool for conducting cancer metastasis-related phosphoproteome research. By optimizing the phosphopeptide enrichment strategy, our data preliminarily clarified the correlation between the abnormality of the phosphoprotein network and lung cancer metastasis. This is expected to be useful for studying phosphorylation sites, phosphorylated proteins, and their signaling pathways related to lung cancer progression.
磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti4+)螯合IMAC材料(Ti4+-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti4+-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti4+-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti4+-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。
Keywords: cancer metastasis; immobilized metal affinity chromatography (IMAC); lung cancer cell; phosphorylation; proteome.