Chromatography peptides

Hemoglobins by Automatic Peptide Chromatography". Cold Spring Harbor Symp. Quant. Biol., (1964), 297. 

Atassi, M. Z., Perlstein, M. T., Rosenblatt, M. C., and Rocek, R, Fully automated simple analytical peptide chromatography on the amino acid analyzer at the 10 8 mole level, Anal. Biochem., 49, 164, 1972. 

Wienkoop, S., Glinski, M., Tanaka, N., Tolstikov, V.V., Fiehn, O., Weckwerth, W. (2004). Linking protein fractionation with multidimensional monolithic reversed-phase peptide chromatography/mass spectrometry enhances protein identification from complex mixtures even in the presence of abundant proteins. Rapid Commun. Mass Spectrom. 18, 643-650. 

The other possibility is to coat the silica with a polymer of defined properties (molecular weight and distribntion) and olefin groups, e.g., polybutadiene, and cross-linked either by radiation or with a radical starter dissolved in the polymer [32]. This method is preferentially used when other carriers like titania and zirconia have to be surface modified. Polyethylenimine has been cross-linked at the snrface with pentaerythrolglycidether [41] to yield phases for protein and peptide chromatography. Polysiloxanes can be thermally bonded to the silica surface. Other technologies developed in coating fnsed silica capillaries in GC (polysiloxanes with SiH bonds) can also be applied to prepare RP for HPLC. 

Another and even more basic problem is protein recovery. This problem is not serious in peptide chromatography. With [125I] angiotensin II (M = 1170 g/mol) RPC was possible on a C 8 column (150 x 4.6 mm, dP = 5 pm) with nearly full recovery at the picogram and femtogram level, but a brandnew column from the same supplier... 

Jones, R. T., Automatic peptide chromatography. Methods Biochem. Anal. 18, 205-258 (1970). 

Peptide chromatography can also be carried out on a reverse-phase column by the ion-pairing technique. The ion pair has increased affinity for the support and allows small hydrophilic peptides to be retained. It also offers a different selectivity and has been reported to improve resolution. Heptane-sulfonic acid and tetrabutylammonium phosphate were used with large peptides (Hancock et al, 1978b), and trifluoroacetic acid was used for both peptides and proteins (Bennett et al, 1979). Amino acid separations have also been carried out on a reverse-phase column using anionic surfactants for ion pairing (Kraak et al, 1977). 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Liquid chromatography is a separation method that is often applied to nonvolatile, thermally labile materials such as peptides, and, if their mass spectra are required after the separation step, then a mild method of ionization is needed. Since FAB/LSIMS is mild and works with a liquid matrix, it is not surprising that attempts were made to utilize this ionization source as both an inlet... 

Reversed-phase high performance Hquid chromatography has come into use for estimating the purity of proteins and peptides as weU. However, before employed, a high performance Hquid chromatographic (hplc) profile of a given protein must be completely vaHdated (43). 

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