HPLC analysis and purification of peptides.

Q4 Biochemistry, Genetics and Molecular Biology Methods in molecular biology Pub Date : 2007-01-01 DOI:10.1007/978-1-59745-430-8_1

Colin T Mant, Yuxin Chen, Zhe Yan, Traian V Popa, James M Kovacs, Janine B Mills, Brian P Tripet, Robert S Hodges

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Abstract

High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic alpha-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins.

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高效液相色谱分析和肽的纯化。

在过去的 25 年中，高效液相色谱法（HPLC）被证明在分离和纯化来源、数量和复杂性千差万别的多肽方面具有极其广泛的用途。本文介绍了用于多肽的主要 HPLC 模式（尺寸排阻、离子交换和反相），并展示了本实验室开发的新型亲水相互作用/阳离子交换混合模式的潜力。除了这些高效液相色谱模式在多肽分离方面的价值外，还将探讨各种高效液相色谱技术在多肽和蛋白质结构表征方面的价值，例如通过尺寸排阻色谱评估多肽/蛋白质的寡聚状态，以及监测两性α-螺旋多肽的亲水性/疏水性，这是开发新型抗菌肽的重要前体。毛细管电泳在多肽分离方面的价值也得到了证明。介绍了多肽和重组蛋白在分析柱和仪器上进行多达 200 毫克样品负载的制备型反相色谱纯化方案。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

2.00

自引率

0.00%

发文量

3536

期刊介绍： For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.