亲水作用-反相二维液相色谱串联质谱法鉴定水稻蛋白质

摘要

建立了亲水作用-反相二维液相色谱串联质谱分析水稻叶片蛋白质组学的方法.利用标准肽段系统分析了液相色谱流动相酸碱度对二维亲水作用-反相色谱系统正交性的影响.结果表明,第一维亲水作用色谱在碱性 (pH 9.3) 和第二维反相色谱在酸性 (pH 3.3) 的条件下,正交性最佳(R2=0.34113).在此基础上,结合馏分收集技术进一步评价了本测试系统在水稻叶片蛋白质分析中的正交性.结果表明,在所有馏分收集组分中,鉴定次数小于2次的水稻叶片肽段占总肽段数目的50%以上,且一维液相色谱馏分收集的肽段在第二维色谱及质谱分离分析中,可以较好地分布在不同时间段的洗脱窗口,表明本研究建立的亲水作用-反相二维液相色谱串联质谱结合馏分收集技术在复杂水稻叶片蛋白质分离鉴定中可提供良好的的分离正交性.结合水稻蛋白质数据库检索,共鉴定出207345个肽段,归属于2930个蛋白质簇.%A two-dimensional liquid chromatography method was developed for the analysis of rice leaves proteomics based on the coupling of hydrophilic interaction liquid chromatography-reversed-phase liquid chromatography with online tandem mass spectrometry. The influence of pH value of chromatographic mobile phase on the orthogonality of the hydrophilic interaction-reversed-phase two-dimensional liquid chromatography was evaluated by the changes of standard peptide retention. The results indicated that the better orthogonality (R2=0.34113) was achieved from the system with hydrophilic interaction columns(pH 9.3) in the first and C18columns(pH 3.3) in the second LC dimension. Coupled with multiple fraction concatenation strategy,the orthogonality of two-dimensional liquid chromatography was further evaluated in the analysis of complex rice leaf proteins. The results showed that more than 50% of the total peptides were identified less than two times, and the peptides obtained from first-dimension were well distributed across the elution window,indicating that the method showed significant orthogonality in the identification of complex rice leaf proteins. Based on the proteome discoverer software,207345 peptides belonged to 2930 protein clusters were identified.