Labeling reactions applicable to chromatography and electrophoresis of minute amounts of proteins

Abstract

Chromatography and electrophoresis have become extremely valuable and important methods for the separation, purification, detection and analysis of biopolymers and HPLC/HPCE may become the premier, preferable approaches for both qualitative and quantitative analyses of most proteins, especially from recombinant materials. This includes smaller peptides, polypeptides, proteins, antibodies and all types of protein or antibody-conjugates (antibody-enzyme, protein-fluorescent probe, antibody-drug and so forth). This entire Topical Issue of Journal of Chromatography emphasizes the application of chromatography and electrophoresis to protein analysis. This particular review deals with approaches to the selective tagging or labeling of proteins at trace (minute) levels, again using either chromatography or electrophoresis, with the emphasis on modern HPLC/HPCE methods and approaches. We discuss here both pre- and post-column labeling methods and reagents, techniques for realizing selective labeling of proteins or antibodies, applicable approaches to protein preconcentration in both HPLC and HPCE areas and in general, methods for improving (lowering) detection limits for proteins utilizing chemical or physical derivatization and/or preconcentration techniques. There are really two major goals or emphases in that which follows: (1) methods for selective labeling of proteins prior to or after HPLC/HPCE and (2) labeling of proteins at trace levels for improved separation-detection and lowered detection limits. We discuss here a large number of specific references related to both pre- and post-column/capillary derivatizations for proteins, as well as methods for improved detectability in both HPLC and HPCE by, for example, analyte preconcentration on a solid-phase extractor or membrane support, capillary isotachophoresis and other methods. Selective reactions or derivatizations on proteins refers to the ability to tag the protein at specific (e.g. reactive amino sites) in a controlled manner, with the products having the same number of tags all at the very same site or sites. The products are all the same species, having the same number of tags at the same locations on the protein. Selective reactions can also refer to the idea of tagging all of the protein sample at only a single, same site or at all available sites, homogeneously.