Rho Jung-hyun, Qin Shuzhen, Wang Julia Y, Roehrl Michael H A
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
J Proteome Res. 2008 Jul;7(7):2959-72. doi: 10.1021/pr8000892. Epub 2008 Jun 13.
Colorectal adenocarcinoma is one of the worldwide leading causes of cancer deaths. Discovery of specific biomarkers for early detection of cancer progression and the identification of underlying pathogenetic mechanisms are important tasks. Global proteomic approaches have thus far been limited by the large dynamic range of molecule concentrations in tissues and the lack of selective enrichment of the low-abundance proteome. We studied paired cancerous and normal clinical tissue specimens from patients with colorectal adenocarcinomas by heparin affinity fractionation enrichment (HAFE) followed by 2-D PAGE and tandem mass spectrometric (MS/MS) identification. Fifty-six proteins were found to be differentially expressed, of which 32 low-abundance proteins were only detectable after heparin affinity enrichment. MS/MS was used to identify 5 selected differentially expressed proteins as proteasome subunit beta type 7 (PSB7), hemoglobin alpha subunit (HBA), peroxiredoxin-1 (PRDX1), argininosuccinate synthase (ASSY), and signal recognition particle 9 kDa protein (SRP9). This is the first proteomic study detecting the differential expression of these proteins in human colorectal cancer tissue. Several of the proteins are functionally related to tissue hypoxia and hypoxic adaptation. The relative specificities of PSB7, PRDX1, and SRP9 overexpression in colon cancer were investigated by Western blot analysis of patients with colon adenocarcinomas and comparison with a control cohort of patients with lung adenocarcinomas. Furthermore, immunohistochemistry on tissue sections was used to define the specific locations of PSB7, PRDX1, and SRP9 up-regulation within heterogeneous primary human tumor tissue. Overexpression of the three proteins was restricted to the neoplastic cancer cell population within the tumors, demonstrating both cytoplasmic and nuclear localization of PSB7 and predominantly cytoplasmic localization of PRDX1 and SRP9. In summary, we describe heparin affinity fractionation enrichment (HAFE) as a prefractionation tool for the study of the human primary tissue proteome and the discovery of PSB7, PRDX1, and SRP9 up-regulation as candidate biomarkers of colon cancer.
结直肠癌是全球癌症死亡的主要原因之一。发现用于早期检测癌症进展的特定生物标志物以及确定潜在的致病机制是重要任务。迄今为止，全球蛋白质组学方法受到组织中分子浓度动态范围大以及低丰度蛋白质组缺乏选择性富集的限制。我们通过肝素亲和分级富集（HAFE），然后进行二维聚丙烯酰胺凝胶电泳（2-D PAGE）和串联质谱（MS/MS）鉴定，研究了结直肠癌患者的配对癌组织和正常临床组织标本。发现56种蛋白质存在差异表达，其中32种低丰度蛋白质仅在肝素亲和富集后才可检测到。利用MS/MS鉴定出5种差异表达蛋白质，分别为蛋白酶体亚基β7型（PSB7）、血红蛋白α亚基（HBA）、过氧化物酶1（PRDX1）、精氨琥珀酸合成酶（ASSY）和信号识别颗粒9 kDa蛋白（SRP9）。这是第一项检测这些蛋白质在人类结直肠癌组织中差异表达的蛋白质组学研究。其中几种蛋白质在功能上与组织缺氧和缺氧适应相关。通过对结肠腺癌患者进行蛋白质免疫印迹分析，并与肺腺癌患者对照队列进行比较，研究了PSB7、PRDX1和SRP9在结肠癌中过表达的相对特异性。此外，利用组织切片免疫组织化学方法确定PSB7、PRDX1和SRP9在异质性原发性人类肿瘤组织中上调的具体位置。这三种蛋白质的过表达仅限于肿瘤内的肿瘤细胞群体，表明PSB7在细胞质和细胞核均有定位，而PRDX1和SRP9主要定位于细胞质。总之，我们将肝素亲和分级富集（HAFE）描述为一种用于研究人类原发性组织蛋白质组的预分级工具，并发现PSB7、PRDX1和SRP9的上调作为结肠癌的候选生物标志物。
