HPLC and CE of Peptides and Proteins

描述（由申请人提供）：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。本实验室主要研究以下四个方面：（1）开发新的多肽和蛋白质的HPLC和毛细管电泳（CE）分离方法;（2）利用反相色谱（RPC）监测多肽和蛋白质的折叠和稳定性;（3）将RPC和亲水相互作用/阳离子交换色谱中的肽洗脱行为相关联（HILIC/CEC）的生物活性;和（4）自动化二维HPLC的蛋白质组学应用中的肽和蛋白质的发展。对肽和蛋白质的有效分析和制备纯化技术的需求不断增加。因此，HPLC在我们的实验室中常规用于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多的治疗重要性的化合物。新的纯化协议，如混合模式HILIC/CEC与挥发性移动的相的发展，将继续竞争RPC。一个普遍适用的一步RPC协议的重组蛋白从全细胞裂解物将代表一个相当大的进步，现有的多步骤的方法。此外，我们的新的样品置换色谱法（SDC）的方法，当按比例缩小到微孔和毛细管RPC柱，将允许有效的纯化，甚至有限的量（0.5毫克至10毫克）的粗合成肽。RPC和HILIC/CEC代表多肽结构特征的有效监测器，并且将有可能将洗脱行为与抗微生物α-螺旋和β-折叠肽的两亲性和亲水性/疏水性相关联以用于SAR（结构活性关系）研究。因此，我们显示了HPLC在了解肽抗微生物机制的作用方面的优势，肽抗微生物机制是重新设计和开发新型抗生素以应对细菌对传统抗生素的耐药性增加的重要环节。蛋白质组学的总体目标是系统地鉴定和定量生物体在特定时间和特定条件下表达的所有蛋白质。蛋白质组学是一个多学科的研究活动，其中分离科学（包括多维HPLC和CE）和质谱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.