Ji H, Whitehead R H, Reid G E, Moritz R L, Ward L D, Simpson R J
Ludwig Institute for Cancer Research, Parkville Victoria, Australia.
Electrophoresis. 1994 Mar-Apr;15(3-4):391-405. doi: 10.1002/elps.1150150158.
Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75-80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (M(r) approximately 18,000, pI 6.7-6.9) and b (M(r) approximately 24,000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (M(r) approximately 66,000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide-mass database.
使用二维电泳凝胶（2-DE）比较了从大肠不同区域分离出的正常人结肠隐窝和几种结肠直肠癌细胞系的蛋白质图谱。通过内在放射性标记和考马斯亮蓝染色检测发现，从结肠升段、降段以及直肠分离出的正常隐窝的蛋白质图谱几乎（>95%）相同。虽然正常隐窝和结肠直肠癌细胞系（LIM 1863，一种在体外可作为类器官生长并自发分化为隐窝样结构的细胞系）中75-80%的蛋白质斑点能够匹配，但大量蛋白质的相对表达水平存在差异。在LIM 1863、LIM 1215和LIM 1899这三种细胞系的2-DE凝胶蛋白质图谱中，检测到至少两个蛋白质斑点（在正常细胞的蛋白质图谱中未检测到），即蛋白质a（相对分子质量约为18,000，等电点6.7 - 6.9）和蛋白质b（相对分子质量约为24,000，等电点5.9 - 6.0）。这些蛋白质的身份尚不清楚，在它们被视为潜在的结肠肿瘤标志物之前还需要进一步研究。LIM 1215细胞是一种具有许多与柱状细胞相关特性的结肠癌细胞系，其约60%的细胞蛋白质能够与LIM 1863细胞匹配。本文展示的结果代表了我们为正常人结肠细胞和几种结肠直肠癌细胞系建立全面蛋白质数据库工作的初始阶段。虽然我们最初的蛋白质鉴定依赖于微测序方法，但我们目前正在评估肽质量指纹图谱技术，利用毛细管反相高效液相色谱（RP-HPLC）和电喷雾质谱，作为在亚皮摩尔水平快速鉴定蛋白质的一种手段。使用这种方法，当针对分子量搜索（MOWSE）肽质量数据库筛选时，从仅七个胰蛋白酶肽质量中就将蛋白质#3（相对分子质量约为66,000，等电点6.2）鉴定为热休克蛋白60。
