以SELDI芯片进行细胞标本蛋白分析的方法学研究

目的:探讨以SELDI芯片技术进行细胞标本蛋白分析的最适方法及条件,筛选细胞标本蛋白表达差异。方法:对细胞标本分别用超声裂解法,U9细胞裂解缓冲液配方和自配细胞裂解液提取蛋白,以BCA法测定蛋白浓度;分别以磁珠活化后点样和生物芯片处理器点样使蛋白样品与芯片结合;并对提取蛋白进行检测,比较不同蛋白浓度梯度点样及WCX2,SAX2,IMAC-Cu,H50芯片捕获蛋白差异,用WCX2芯片筛选蛋白差异表达。结果:相同培养条件细胞以上述三种不同蛋白提取方法获得的蛋白浓度分别为:0.25±0.034μg/μl,0.6±0.06μg/μl,1.02±0.077μg/μl;生物芯片处理器点样法操作简单,要求样本量较少,点样时间短;SELDI芯片蛋白质峰图谱与蛋白浓度呈较好的正相关;WCX2,SAX2,H50,IMAC-Cu芯片捕获的蛋白质种类有较大区别;在分子量1000～300000Da范围内,以WCX2芯片共检测到87个差异蛋白峰,其中17个呈趋势变化。结论:上述三种方法比较,选用自配的细胞裂解液提取蛋白的浓度较高且更适于芯片研究;生物芯片处理器能较好地使蛋白与芯片结合;SELDI芯片能准确定位蛋白,且其蛋白质峰与被测蛋白浓度呈正相关变化;SELDI各芯片捕获蛋白类型不同,选择适宜芯片或联合运用芯片检测更易获得较理想蛋白差异表达结果。

A Study of SELDI-TOF-MS Protocol for Analyzing Proteins from Cells

Objective:To study the optimum protocol for analyzing proteins from cells by SELDI-TOF-MS.Methods:Proteins were extracted from cell samples by ultrasonic disruption, U9 cell disruption buffer and cell disruption buffer were established respectively. The concentrations of proteins were detected by BCA method, then protein samples were applied directly to the ProteinChips arrays using WCX magnetic bead and the bioprocessor, respectively, and protein samples were captured by Chips of WCX2, SAX2, IMAC Cu and H50, respectively, their results were compared, and detailed protein expression differences were detected using the chip of WCX2.Results:The concentrations of proteins isolated from cells using 3 different ways are: 0.25±0.034μg/μl，0.6±0.06μg/μl，1.02±0.077μg/μl,respectively. The method of Biochip Processor is simple, which requires less sample size and time. There is a good relationship between the protein peaks and protein concentrations in SELDI protein spectra. There are differences in the type of proteins captured by chips of WCX2, SAX2, H50, IMAC-Cu. In the limit of 1000~300 000Da, 87 protein expression difference peaks were detected by WCX2 chip, amount of which, 17 show a tendency variation.Conclusion:Using self prepared cell disruption buffer receive highest protein concentration and which is better for further test.Bio-Chip Processor does better in application of sample.SELDI can not only detect protein expression differences but also reflect the protein concentration relationship; Selecting an appropriate chip or combining use of different chips to detect the protein expression differences can obtain more satisfactory results.