HPLC and CE of Peptides and Proteins

描述(由申请人提供)：高效高效液相色谱(HPLC)方案的持续发展对生物医学科学和生物技术的所有领域都至关重要。我们实验室正在研究的四个主要领域是：(1)开发新的多肽和蛋白质的高效液相色谱和毛细管电泳色谱分离方法；(2)反相色谱(RPC)用于监测多肽和蛋白质的折叠和稳定性；(3)在RPC和亲水相互作用/阳离子交换色谱(HILIC/CEC)中的多肽洗脱行为与生物活性之间的关联；以及(4)在蛋白质组学应用中多肽和蛋白质的自动化二维高效液相色谱的开发。对多肽和蛋白质的高效分析和制备纯化技术的需求不断增加。因此，我们实验室经常使用高效液相色谱法来纯化天然蛋白质和蛋白质片段、重组蛋白质和合成肽，后者是一类具有越来越重要治疗作用的化合物。新的净化协议，如混合模式HILIC/CEC和挥发性流动相的开发，将继续与RPC竞争。一种普遍适用的从全细胞裂解物中提取重组蛋白的一步RPC方法将代表着现有多步方法的相当大的进步。此外，我们新的样品置换色谱(SDC)方法，当缩小到微孔和毛细管RPC柱时，将允许高效纯化即使是有限数量(0.5毫克到10毫克)的粗合成肽。RPC和HILIC/CEC都是多肽结构特征的有效监测器，并且有可能将洗脱行为与抗微生物α-螺旋和β-折叠多肽的两亲性和亲水性/疏水性相关联，用于SAR(结构活性关系)研究。因此，我们展示了高效液相色谱法在理解多肽抗菌机制的作用方面的优势，这是从头设计和开发新型抗生素的关键环节，以应对细菌对传统抗生素日益增长的耐药性。蛋白质组学的总体目标是对生物体在特定时间和特定条件下表达的所有蛋白质进行系统的鉴定和量化。蛋白质组学是一项多学科的研究活动，其中分离科学(包括多维高效液相色谱和毛细管电泳法)和质谱学将继续发挥关键作用。

DESCRIPTION (provided by applicant): The continuing development of efficient high-performance liquid chromatography (HPLC) protocols is critical for all areas of biomedical science and biotechnology. The four major areas under investigation in our laboratory are: (1) development of novel HPLC and capillary electrophoresis (CE) separation methods for peptides and proteins; (2) utility of reversed-phase chromatography (RPC) to monitor folding and stability of peptides and proteins; (3) to correlate peptide elution behavior in RPC and hydrophilic interaction/cationexchange chromatography (HILIC/CEC) with biological activity; and (4) development of automated two dimensional HPLC for peptides and proteins in proteomics applications. There is an ever-increasing demand for efficient analytical and preparative purification techniques for peptides and proteins. Thus, HPLC is used routinely in our laboratory for purification of native proteins and protein fragments, recombinant proteins and synthetic peptides, the latter of which are a class of compounds with increasing therapeutic importance. Novel purification protocols, such as mixed-mode HILIC/CEC with the development of volatile mobile phases, will continue to rival RPC. A generally applicable one-step RPC protocol for recombinant proteins from whole cell lysates will represent a considerable advance on existing multi-step approaches. Also, our novel sample displacement chromatography (SDC) approach, when scaled down to microbore and capillary RPC columns, will allow efficient purification of even limited amounts (0.5 mg to 10 mg) of crude synthetic peptides. Both RPC and HILIC/CEC represent potent monitors of polypeptide structural characteristics and it will be possible to correlate elution behavior with amphipathicity and hydrophilicity/hydrophobicity of antimicrobial alpha-helical and beta-sheet peptides for SAR (structure activity relationships) studies. Thus, we are showing the advantages of HPLC in the understanding of the action of peptide antimicrobial mechanisms, a vital link in the de novo design and development of novel antibiotics as a response to the increasing bacterial resistance to traditional antibiotics. The overall aim of proteomics is the systematic identification and quantification of all proteins expressed by an organism at a certain time and under certain conditions. Proteomics is a multidisciplinary research activity wherein separation science (including multidimensional HPLC and CE) and mass spectrometry will continue to play pivotal roles.