Cation-exchange chromatography protein separation

Weitzhandler, M., Farnan, D., Horvath, J., Rohrer, J. S., Slingsby, R. W., Avdal-ovic, N., and Pohl, C., Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases, /. Chromatogr. A, 828, 365, 1998. 

It was in this context that the first true comprehensive online LC x LC separation was reported (Bushey and Jorgenson, 1990). Mixtures of intact proteins were analyzed using cation-exchange chromatography (CEX) as the first dimension and size exclusion chromatography (SEC) as the second. This research demonstrated that the practical difficulties of coupling two dissimilar LC modes for a comprehensive 2D separation are relatively easy to overcome when instrumentation is properly configured. 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

In addition to classical reverse phase separation of peptides on octadecyl derivatized silica monoliths, sugars and peptides as well as proteins and nucleosides have been analyzed on a 20-cm-long silica-based poly(acrylic acid) column (ID. 200 pm), employing HILIC and weak cation-exchange chromatography, respectively [194]. Furthermore, HILIC fractionation of polysaccharides delivered remarkable and promising results [84,194]. 

CM Hollar, AJR Law, DG Dagleish, RJ Brown. Separation of major casein fractions using cation-exchange fast protein liquid chromatography. J Dairy Sci 74 2403-2409, 1991. 

In the gel-free approaches, also referred as shotgun proteomics, proteins in a mixture are digested and the peptides released separated by reverse-phase HPLC, either alone or combined with strong cation exchange chromatography... 

The hexapeptide leader sequence was removed from the fusion protein by incubation in 50 mM Tris/HCl buffer, pH 8.0, with Endoproteinase Arg-C (1 100, enzymeisubstrate, w/w), for 3 h at 37°C or overnight at 37°C (1 600, enzyme substrate, w/w). Cleavage can be monitored by analytical reverse phase HPLC, where a small decrease in retention time is observed for the cleaved product (Fig. 4). The appearance of peptides with low retention times resulting from cleavage of the MKKKWPR sequence are observed. The cleaved product is separated by cation exchange chromatography as described in Subheading... 

The bottom-up approach differs in that the entire proteome is first digested with trypsin and then the fragments are separated by cation-exchange chromatography followed by reverse phase chromatography84,124. The fragments are then analyzed by mass spectrometry and are used to identify the proteins in the sample. 

The process of separation and quantitation of amino acids has been automated. In one automated method, a single cation exchange resin column separates all the amino acids in the protein hydrolysate. The analyzer is capable of detecting as little as 1-2 nmol of an amino acid and a complete analysis can be obtained in about 4 hours. In newer procedures, the complete analysis can be performed in about Ihour and permit detection of as little as 1-2 nmol of an amino acid. Picomole amounts of amino acids can be determined when the separated amino acids are coupled to fluorescent reagents such as o-phthalaldehyde. Amino acid separation and quantitation can also be accomplished by reverse-phase high-pressure liquid chromatography of amino acid derivatives—a rapid and sensitive procedure. 

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