范式转变:依赖离子配对的大小排除效应在自下而上的蛋白质组学逆相分离中的主要作用
Darien Yeung1, Victor Spicer2, Rene P Zahedi1,2,3,4
1Department of Biochemistry and Medical Genetics, University of Manitoba, 336 BMSB, 745 Bannatyne Avenue, Winnipeg R3E 0J9, Canada.
Analytical chemistry
|May 29, 2024
概括
对的逆相色谱保留率并不总是更高,更大的表面积或孔径大小. 酸化物显示在200 Å的峰值保留,挑战了蛋白质组学中常见的假设.
科学领域:
- 分析化学 分析化学
- 染色体学 染色体学 是一种染色学.
- 蛋白质组学是指蛋白质组学.
背景情况:
- 在逆相高性能液态染色学 (RP-HPLC) 中,常见的智慧假定表面积和孔径较大时的保留率更高.
- 现有的保留模型往往不考虑由化物组成,特别是离子配对剂影响的特定相互作用.
研究的目的:
- 为了研究柱孔大小和化剂成分 (三酸与酸) 对保留和分离选择性的影响.
- 挑战和完善关于蛋白质学应用的RP-HPLC表面积和毛孔大小效应的基本假设.
主要方法:
- 使用100和120 Å完全多孔C18吸收剂的分离选择性的比较.
- 系统评估逆相高性能液态染色学保留在20个柱子的孔径大小从60到300 Å.
- 使用酸 (FA) 和三酸 (TFA) 基化物进行分析.
主要成果:
- 对于具有较小表面积的较宽孔包装材料,观察到意想不到的更高的保持率.
- 的保留增加,孔径大小高达200 Å的FA化剂和120-200 Å的TFA化剂,在FA中狭孔颗粒的保留更明显下降.
- 用FA和TFA溶解剂形成的离子对的大小显著影响的保留和选择性,特别是对于较小的.
结论:
- 关于分析物大小和可访问表面积的常见假设需要对蛋白质组学中小的离子对RP分离进行重新评估.
- 基于的混合材料,孔径为130-200 Å,建议用于自下而上的蛋白质应用,以优化峰值形状和保持性.
- 加强LC列制造商和蛋白质LC专家之间的协作对于开发蛋白质组学最佳RPLC列至关重要.
相关概念视频
01:08Size-Exclusion Chromatography
560
In size-exclusion chromatography (SEC), also known as molecular-exclusion or gel-permeation chromatography, molecules are separated based on their sizes. This technique is important for separating large molecules such as polymers and biomolecules. The two classes of micron-sized stationary phases encountered in SEC are silica particles and cross-linked polymer resin beads. Both materials are porous, but their pore sizes vary significantly.
Silica particles offer advantages such as rigidity,...
Silica particles offer advantages such as rigidity,...
560
01:17Ion Exchange
583
Ion exchange chromatography separates charged molecules from a solution by reversibly exchanging them with mobile, or 'active', ions associated with the oppositely charged stationary phase. This method can be used to separate ions, soften and deionize water, and purify solutions. The polymers comprising the ion-exchange column are high-molecular-weight and chemically stable polymers, crosslinked to be porous and essentially insoluble. They are also functionalized with either acidic or...
583
01:30Capillary Electrophoresis: Applications
389
Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...
389
01:27SDS-PAGE
27.8K
Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...
A variation of gel electrophoresis, termed polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact...
27.8K