HPLC OF PEPTIDES AND PROTEINS

研究的三个主要领域是：（1）开发新的肽/蛋白质分离方法;（2）通过样品置换色谱法（SDC）进行多重和同时的肽纯化;以及（3）利用反相色谱法（RPC）监测肽的稳定性、折叠和构象。 对合成肽的不断增长的需求推动了对有效的分析和制备纯化技术的持续需求。 新的纯化方案，如混合模式亲水相互作用/阳离子交换色谱（HILIC/CEC）与挥发性移动的相的发展，将竞争对手RPC。 定制的RPC固定相将允许肽混合物的洗脱模式的有利操纵。 多个肽合成的要求将得到满足的快速，低压和同时纯化技术的基础上结合的固相萃取技术与新的SDC方法的发展，允许多达100肽的时间被纯化的有机改性剂的情况下。样品置换技术与微孔RPC的组合以在直接洗脱到LC-MS中之前分离和浓缩合成杂质，也将允许比现有方法的灵敏度增加至少100倍以判断肽同质性。 RPC是多肽结构特征的有效探针，并且将有可能将洗脱行为与抗微生物α-螺旋和环状β-折叠肽的两亲性和疏水性相关联。 这些工作对重新设计更有效的抗菌剂具有重要意义。

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.