HPLC OF PEPTIDES AND PROTEINS

研究的三个主要领域是：(1)开发新的多肽/蛋白质分离方法；(2)通过样品置换色谱(SDC)进行多肽同时纯化；(3)反相色谱(RPC)用于监测多肽的稳定性、折叠和构象。对合成肽不断增长的需求推动了对高效分析和制备纯化技术的持续需求。随着挥发性流动相的发展，新的纯化方案，如混合模式亲水相互作用/阳离子交换层析(HILIC/CEC)，将与RPC竞争。定制的RPC固定相将允许对多肽混合物的洗脱模式进行有利的操作。基于固相萃取技术和新的SDC方法的结合，一种快速、低压和同时纯化技术的开发将满足多肽合成的要求，在没有有机修饰剂的情况下，允许一次纯化多达100个肽。将样品置换技术与微孔RPC相结合，在直接洗脱到LC-MS之前分离和浓缩合成杂质，也将使判断多肽均一性的现有方法的灵敏度至少提高100倍。RPC是多肽结构特征的有效探针，它将有可能将洗脱行为与抗菌α-螺旋和环β-片肽的两亲性和疏水性相关联。这项工作对更有效的抗菌剂的从头设计具有重大意义。

The three major areas under investigation are: (1) development of novel separation methods of peptides/proteins; (2) multiple and simultaneous peptide purification by sample displacement chromatography (SDC); and (3) utility of reversed-phase chromatography (RPC) to monitor stability, folding and conformation of peptides. The ever-increasing demand for synthetic peptides has fueled a constant demand for efficient analytical and preparative purification techniques. Novel purification protocols, such as mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEC) with the development of volatile mobile phases, will rival RPC. Tailored RPC stationary phases will allow the advantageous manipulation of the elution patterns of peptide mixtures. A requirement for multiple peptide synthesis will be met by the development of a rapid, low pressure and simultaneous purification technique based on a combination of solid-phase extraction technology coupled with the novel SDC approach, allowing up to 100 peptides at a time to be purified in the absence of organic modifier. Combination of the sample displacement technique with microbore RPC to separate and concentrate synthetic impurities prior to direct elution into LC-MS will also allow at least a 100-fold increase in sensitivity over present methods to judge peptide homogeneity. RPC is a potent probe of polypeptide structural characteristics and it will be possible to correlate elution behaviour with amphipathicity and hydrophobicity of antimicrobial alpha-helical and cyclic beta-sheet peptides. Such work has major implications for the de novo design of more effective antimicrobial agents.