Peptides separation

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

Grossman, R D., Wilson, K. J., Petrie, G., and Lauer, H. H., Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis, Anal. Biochem., 173, 265, 1988. 

The orthogonality of common LC modes for peptide separation is not well known, but in most cases it is not ideal. Several reports suggest that even dissimilar modes such as SCX and RP do not separate peptides in an orthogonal fashion, and some peak... 

TABLE 12.1 Chromatographic Modes and Conditions Used for LC-MS Study of Tryptic Peptides Separation Selectivity... 

Young, P.M., Wheat, T.E. (1990). Optimization of high-performance liquid chromatographic peptide separations with alternative mobile and stationary phases. J. Chromatogr. 512,273-281. 

TWO-DIMENSIONAL HPLC-CE METHODS FOR PROTEIN/PEPTIDE SEPARATION... 

J. Gao, J.D. Xu, L.E. Locascio, and C.S. Lee, Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal. Chem. 73, 2648-2655 (2001). 

For reproducible expression analysis and protein quantification MS methods based on isotopic labeling are available. They were designed in conjunction with two or more dimensional chromatographic peptide separation coupled online to MS and require advanced bioinformatics input to analyze the complex data sets in a reasonable time frame. This is also true for the alternative fluorescence-based technology of differential gel electrophoresis (DIGE Fig. 10.6) with tailor-made software which allows statistical validation of multiple data sets. 

Protease cleaves peptide, separates quencher and biotin... 

It is too early to draw any conclusions about the insensitivity of the rate constants to the nature of the dipeptide. Differences among the peptides seem to be revealed more in the temperature dependencies of the rate constants for intramolecular electron transfer than in the magnitude of the rate constant itself. Work is in progress on the synthesis of other di-, tri-, and tetra-peptides separating Co(III) and Ru(II) in order to examine the temperature dependence of the intramolecular rate... 

---