Protein pl shifts due to posttranslational modifications in the separation and characterization of proteins
Protein pl shifts due to posttranslational modifications in the separation and characterization of proteins
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DOI：
10.1021/ac048494w
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发表时间：
2005-05-01
影响因子：
7.4
通讯作者：
Barder, TJ
中科院分区：
文献类型：
--
作者：
Zhu, K;Zhao, J;Barder, TJ
使用2-D液体分离技术表征来自乳腺癌细胞系的蛋白质，其中蛋白质pI用作第一维分离参数。为了实现这种蛋白质pI分离，采用色谱聚焦（CF），其中使用弱阴离子交换介质在柱上产生pH梯度，其中每0.2 pH单位分级分离并收集完整蛋白质。它表明，表达的完整蛋白质的pI产生的CF是一个重要的参数，用于识别和表征的实际发生在癌细胞中的蛋白质修饰。对于大多数蛋白质，实验测定的pI非常接近数据库预测的pI。然而，在其他情况下，当观察到pI从预期值偏移时，表明这种偏移通常与蛋白质修饰相关。引起这种变化的修饰包括在癌细胞中经常观察到的截短和缺失或可使pI变化几个pH单位的磷酸化。它还表明，磷酸化对所观察到的移位的影响可以根据蛋白质和磷酸化的量而变化。此外，在磷酸化后，对于pI高于7.0的蛋白质，经常观察到pI的大的变化，而对于pI接近5.0的蛋白质，观察到的变化很小。因此，表达蛋白的pI值与完整MW值、肽图谱和MS/MS结果一起成为鉴定癌细胞中发生的翻译后修饰的存在和类型的重要参数。
Proteins from breast cancer cell lines are characterized using a 2-D liquid separation technique in which protein pI is used as the first-dimension separation parameter. To effect this protein pI separation, chromatofocusing(CF) is employed whereby a pH gradient is generated on-column using a weak anion exchange medium with the intact proteins fractionated and collected every 0.2 pH unit. It is demonstrated that the pI for expressed intact proteins as generated by CF is an important parameter for identification and characterization of the actual protein modifications occurring in the cancer cell. For most proteins, the experimentally determined pI is very close to that predicted by the databases. In other cases, however, where the pI is observed to be shifted from the expected value, it is shown that this shift is often correlated to protein modifications. The modifications that cause such shifts include truncations and deletions often observed in cancer cells or phosphorylations that can shift the pI by several pH units. It is also shown that the effects of phosphorylation on the observed shift can vary depending upon the protein and the amount of phosphorlyation. Moreover, large changes in the pI are often observed for proteins with a pI above 7.0 upon phosphorlyation, whereas little change is observed for proteins with a pI of similar to 5.0. The expressed protein's pI value thus becomes an important parameter together with the intact MW value, peptide map, and MS/MS results for identification of the presence and type of posttranslational modifications occurring in the cancer cell.