Separation of proteins, peptides, and amino

TABLE II. Improvements on the Capillary Electrophoresis Separation of Proteins, Peptides and Amino Acids by Deactivation of the Silica Surface or by Addition of Chemical Agents to the Separation Buffer... 

Jan Moebius, Rudolf-Virchow-Center for Experimental Biomedicine, Wurzburg, Germany, Chromatography-Based Separation of Proteins, Peptides, and Amino Acids... 

Hyflo Super Cel has been used by Gardell et used asbestos or glass wool in separations of proteins, peptides, and amino acids. 

Although numerous examples of capillary electrophoresis separations of micro- and macromolecules can be cited (3-9), the most troublesome (and probably the application most commonly used), is the separation and analysis of proteins, peptides and amino acids. Table I shows a comprehensive view of the literature regarding the analysis of these substances, which are biologically the most diverse of all biological compounds, serving a vast array of functions. 

The A. B. Pharmacia brochure on Sephadex in gel filtration listed about 100 references in the period 1959-62 on the use of Sephadex in the isolation, separation, and purification of proteins, peptides, and amino acids. Brochure No. 1, 1965, lists 638 references and gives cross references to abstract cards. 

Isoelectric focussing allows the separation of zwitterionic analytes such as proteins or peptides according to their isoelectric point, pi (section 1.1.1.1). lEF is applied to the separation and purification of proteins, peptides and amino acids on an analytical as well as preparative scale. The pi of a protein depends on the sum of all charges, as well as the 3D structure and post translational modiflcations such as phosphorylation, glycosilation and changes in oxidation state. The pi is, thus, a valuable parameter for studying post-translational modifications of proteins. 

Tim Wehr is Staff Scientist at Bio-Rad Laboratories in Hercules, California. He has more than 20 years of experience in biomolecule separations, including development of HPLC and capillary electrophoresis methods and instrumentation for separation of proteins, peptides, amino acids, and nucleic acids. He has also worked on development and validation of LC-MS methods for small molecules and biopharmaceuticals. He holds a B.S. degree from Whitman College, Walla Walla, Washington, and earned his Ph.D. from Oregon State University in Corvallis. 

This approach has been applied to the separation of polymers, oligomers, and monomers. Each fraction is then farther separated using a suitable separation method. One example is the separation of saccharides in exploded wood. Proteins, peptides, and amino acids were analyzed, step by step, and an automated column-switching separation was studied. However, the separation of proteins does not compete with that achieved by two-dimensional electrophoresis. 

Hearn, M.T., M.I. AgnUar, T. Nguyen, and M. Fridman, High-performance liquid chromatography of amino acids, peptides and proteins. LXXXTV. Application of derivative spectroscopy to the study of column residency effects in the reversed-phase and size-exclusion liquid chromatographic separation of proteins. J Chromatogr, 1988. 435(2) 271-84. 

The formation of methyl- (MTH) or phenylthiohydantoin (PTH) amino acids is a valuable technique for sequencing of amino acids in peptides and proteins by the Edman degradation procedure [1], HPLC is very useful for the separation of MTH- or PTH-amino acids as adsorption [2,3], reversed-phase [4] and ion-exchange [S] chromatography. 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

Separation of proteins based on differences in their electrical charge depends on their acid-base properties, which are mostly determined by the number and type of ionizable side groups in the peptide chain. Since proteins are different from each other with respect to their composition and amino acid sequence, they also have distinct acid-base properties. Information on these properties allows a prediction of the behavior of a given protein when exposed to an electrical field. 

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