Protein/peptide analysis chromatography

Murphy, R.E. (2001). Comprehensive two-dimensional liquid chromatography for comparative protein analysis. Presented at the International Symposium on the Separation of Proteins, Peptides and Polynucleotides. (ISPPP 2001) Orlando, FL. 

Janini, G.M., Chan, K.C., Conrads, T.P., Issaq, H.J., Veenstra, T.D. (2004). Two-dimensional liquid chromatography-capillary zone electrophoresis—sheathless electrospray ionization-mass spectrometry evaluation for peptide analysis and protein identification. Electrophoresis 25, 1973-1980. 

Due to their defined monomodal macropore distribution (see Section 1.2.1), monolithic stationary phases, based on polymerization of organic precursors, are predestined for efficient and swift separation of macromolecules, like proteins, peptides, or nucleic acids, as their open-pore structure of account for enhanced mass transfer due to convection rather than diffusion. In fact, most of the applications of organic monolith introduced and investigated in literature are directed to analysis of biomolecule chromatography [29]. 

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

C. T. Mant, R. S. Hodges, in High-Performance Liquid Chromatography of Peptides and Proteins Separation, Analysis, and Conformation, C. T. Mant, R. S. Hodges (Eds.), CRC Press, Boca Raton, FL, 1991, p. 125. 

FAB and PD have been replaced by electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) in the analytical mass spectrometry laboratory, because both of these newer techniques have a wider mass range of analysis and have lower detection limits. ESI and MALDI have become invaluable ionization techniques for nonvolatile components. This is particularly true for a wide range of biological molecules including proteins, peptides, nucleic acids, etc. Samples can be analyzed by ESI using either direct injection or introduction through liquid chromatography. 

Merritt, C., Jr., and Robertson, D. H. (1967). The analysis of proteins, peptides and amino acids by pyrolysis-gas chromatography and mass spectrometry, f. Gas Chromatogr. 5 96—98. 

Zavitsanos, P., and Goetz, H. (1991). The practical application of diode array UV-visible detectors to high-performance liquid chromatography analysis of peptides and proteins. In High-Performance Liquid Chromatography of Peptides and Proteins Separation, Analysis,... 

Another important development in the field of biopolymer analysis is the introduction of matrix-assisted laser desorption ionization (MALDl), which is a rather recent soft ionization technique that produces molecular ions of large organic molecules. In combination with time-of-flight (TOP) mass spectrometry, it was proposed as a valuable tool for the detection and characterization of biopolymers, such as proteins, peptides, and oligosaccharides, in many types of samples.The use of these recently developed techniques has not decreased the use of chromatography in determinations of biopolymers. Some efforts on the adaptation of the separation abilities of HPLC to the high potential of MALDl-TOF for the sensitive determination of additives in biocomposites are currently being carried out. 

A comprehensive proteomics approach has been developed to identify the components of phosphoprotein complexes and includes the following experimental steps isolation of native phosphoproteins and associated proteins by affinity chromatography, ID PAGE separation of the affinity-isolated proteins, proteolysis of the protein spots, LC-ESI-MS/MS analysis of the cleaved peptides, and database searching for indentification of the proteins involved in the complex formation.178... 

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