Ji Chengjie, Li Laiji, Gebre Mulu, Pasdar Manijeh, Li Liang
Departments of Chemistry and Cell Biology, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
J Proteome Res. 2005 Jul-Aug;4(4):1419-26. doi: 10.1021/pr050094h.
A strategy based on isotope labeling of peptides and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry (LC-MALDI MS) has been employed to accurately quantify and confidently identify differentially expressed proteins between an E-cadherin-deficient human carcinoma cell line (SCC9) and its transfectants expressing E-cadherin (SCC9-E). Proteins extracted from each cell line were tryptically digested and the resultant peptides were labeled individually with either d(0)- or d(2)-formaldehyde. The labeled peptides were combined and the peptide mixture was separated and fractionated by a strong cation exchange (SCX) column. Peptides from each SCX fraction were further separated by a microbore reversed-phase (RP) LC column. The effluents were then directly spotted onto a MALDI target using a heated droplet LC-MALDI interface. After mixing with a MALDI matrix, individual sample spots were analyzed by MALDI quadrupole time-of-flight MS, using an initial MS scan to quantify the dimethyl labeled peptide pairs. MS/MS analysis was then carried out on the peptide pairs having relative peak intensity changes of greater than 2-fold. The MS/MS spectra were subjected to database searching for protein identification. The search results were further confirmed by comparing the MS/MS spectra of the peptide pairs. Using this strategy, we detected and compared relative peak intensity changes of 5480 peptide pairs. Among them, 320 peptide pairs showed changes of greater than 2-fold. MS/MS analysis of these changing pairs led to the identification of 49 differentially expressed proteins between the parental SCC9 cells and SCC9-E transfectants. These proteins were determined to be involved in different pathways regulating cytoskeletal organization, cell adhesion, epithelial polarity, and cell proliferation. The changes in protein expression were consistent with increased cell-cell and cell-matrix adhesion and decreased proliferation in SCC9-E cells, in line with E-cadherin tumor suppressor activity. Finally, the accuracy of the MS quantification and subcellular localization for 6 differentially expressed proteins were validated by immunoblotting and immunofluorescence assays.
一种基于肽段同位素标记和液相色谱-基质辅助激光解吸电离质谱（LC-MALDI MS）的策略已被用于准确量化并可靠鉴定E-钙黏蛋白缺陷型人癌细胞系（SCC9）与其表达E-钙黏蛋白的转染细胞系（SCC9-E）之间差异表达的蛋白质。从每个细胞系中提取的蛋白质用胰蛋白酶消化，所得肽段分别用d(0)-或d(2)-甲醛进行标记。将标记后的肽段混合，肽段混合物通过强阳离子交换（SCX）柱进行分离和分级。每个SCX级分中的肽段再通过微径反相（RP）液相色谱柱进一步分离。然后使用加热液滴LC-MALDI接口将流出物直接点样到MALDI靶板上。与MALDI基质混合后，通过MALDI四极杆飞行时间质谱对单个样品点进行分析，利用初始质谱扫描对二甲基标记的肽段对进行定量。然后对相对峰强度变化大于2倍的肽段对进行串联质谱（MS/MS）分析。将MS/MS谱图进行数据库搜索以鉴定蛋白质。通过比较肽段对的MS/MS谱图进一步确认搜索结果。使用该策略，我们检测并比较了5480个肽段对的相对峰强度变化。其中，320个肽段对的变化大于2倍。对这些变化的肽段对进行MS/MS分析，鉴定出亲本SCC9细胞和SCC9-E转染细胞系之间49个差异表达的蛋白质。这些蛋白质被确定参与调节细胞骨架组织、细胞黏附、上皮极性和细胞增殖的不同途径。蛋白质表达的变化与SCC9-E细胞中细胞间和细胞与基质黏附增加以及增殖减少一致，符合E-钙黏蛋白的肿瘤抑制活性。最后，通过免疫印迹和免疫荧光测定验证了6个差异表达蛋白质的质谱定量和亚细胞定位的准确性。
