Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer.
Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer.
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DOI：
10.1371/journal.pone.0082589
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Jenster G
中科院分区：
文献类型：
--
作者：
Duijvesz D;Burnum-Johnson KE;Gritsenko MA;Hoogland AM;Vredenbregt-van den Berg MS;Willemsen R;Luider T;Paša-Tolić L;Jenster G
目前前列腺癌的标志物，如PSA缺乏特异性。因此，需要新的生物标志物。不幸的是，体液的复杂性往往阻碍了生物标记物的发现。一种有吸引力的替代方法是分离小囊泡，即外切体，∼100 nm，其中包含组织特有的蛋白质，因此可以被认为是疾病特异性生物标记物发现的宝库。用超速离心法从2个永生化的原代前列腺上皮细胞(PNT2C2和RWPE-1)和2个前列腺癌细胞系(PC346C和VCaP)中分离到外切体。在胰酶消化后，蛋白质组学分析使用了纳米LC和串联MS(MS/MS)模式下运行的LTQ-Orbitrap。采用精确质量和时间(AMT)标记法对多肽进行鉴定和定量。采用免疫印迹和免疫组织化学方法对候选生物标志物进行验证。蛋白质组学分析表明，PNT2C2、RWPE-1、PC346C和VCaP的外体中至少含有2个多肽，分别鉴定出248、233、169和216个蛋白质。统计分析表明，PCa和对照细胞之间有52种蛋白质含量不同，其中9种蛋白质在PCa中含量更高。经Western blotting验证，在PCa外体中存在较高丰度的FASN、XPO1和PDCD6IP(Alix)。利用高效LC-FTMS鉴定外体蛋白，发现了PDCD6IP、FASN、XPO1和ENO1作为前列腺癌新的候选生物标志物。
Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ∼100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery. Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry. Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer.
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