蛋白质分离和鉴定的新技术新方法研究进展
Recent advances of technique and method on the protein separation and identification in the proteomic studies
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摘要: 综述了国家重大科学研究计划"蛋白质分离和鉴定新技术新方法"课题的研究进展:高丰度蛋白质去除方面,采用强阳离子交换色谱(SCX)和反相色谱(RPLC),构建二维液相色谱分离系统;利用分子印迹技术制备高丰度蛋白质印迹聚合物.低丰度蛋白分离富集方面,采用新型功能材料磷酸化蛋白质/多肽、糖基化蛋白质/多肽、蛋白质/多肽实现对低丰度蛋白质的选择性富集.多维、多模式、阵列式的蛋白质高效分离方面,利用循环分离体积排阻色谱(csrSEC)和RPLC的联用构建多维分离系统;构建弱阴弱阳离子混合色谱-固定化酶反应器-RPLC-电喷雾质谱系统.高灵敏鉴定新技术新方法方面,发展液质联用接口技术,以中空纤维膜为根本,设计以此为核心的集成化样品预处理装置;新发展多肽衍生试剂,能够将肽段在质谱的检测灵敏度提高1-2个数量级;在靶体材料方面,采用复合核壳纳米功能靶体材料;在质谱数据处理方法方面,基于遗传算法发展筛选标准优化策略.这些新技术和新方法为蛋白质组研究提供了有效的方法.选择性吸附材料的研制为蛋白质组学研究中高丰度蛋白质去除和低丰度蛋白质富集提供了新的途径,进而避免了复杂体系对目标蛋白质鉴定的干扰.以多维、多模式、阵列式的蛋白质高效分离技术为核心构建的平台,实现了蛋白质组的高效、高通量、高可靠性分离;以液相色谱/质谱联用为核心的高灵敏鉴定的新技术新方法的发展,提高了低丰度蛋白质的质谱鉴定灵敏度和准确度.Abstract: The new techniques and methods of protein separation and identification in the program "Novel Techniques and Methods for Protein Separation and Identification",supported by "National Basic Research Program of China",were reviewed.For high-abundance proteins depletion,strong cation exchange (SCX) chromatography was coupled with reversed phase liquid chromatography (RPLC) to develop two dimensional liquid chromatography,and high abundance proteins were applied as the templates to developed molecularly imprinted materials.For low abundance protein enrichment selectively,novel functional materials were developed for selectively capturing significant post-translated proteins,such as phosphopeptides/proteins and glycopeptides/proteins.For multidimensional,multi-mode and array protein separation,column switch recycling size exclusion chromatography (csrSEC) was coupled with RPLC to develop multidimensional liquid chromatography,and weak anion and cation exchange chromatography mixed-bed microcolumn were integrated with the immobilized trypsin reactor and RPLC-ESI/MS/MS to develop at high-throughput proteome platform.During the study of high-sensitive identification techniques,an online integrated platform for sample pretreatment,which was based on the hollow fiber membrane interface for solvent exchange,was established.New reagents were developed for the peptide derivatization,which decreased the limit of detection of peptide on the mass spectrum by 10-100 times.Core-shell nanoparticles were employed on the plate materials directly for the matrix-assisted laser desorption/ionization mass (MALDI).A predictive genetic algorithm was implemented for the optimization of filtering criteria to maximize the number of identified peptides for mass spectrum database searching.The new technology and new method for proteomic study provides an effective method.The development of selective adsorption materials provides a new way for high abundance protein depletion and low abundance proteins enrichment of proteomics research,and avoids the interference of the target protein identification in complex system.The platform of multidimensional and mode,array type of protein efficient separation technology as the core is constructed and realized the protein group of high efficiency,high flux,high reliability separation.With the new technology and new method development of high performance liquid chromatography/mass spectrometry as the core of high sensitive identification,the sensitivity and accuracy of low abundance protein mass spectrum identification are improved.
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