0000000000512113
AUTHOR
Lars-erik Edholm
Protein mapping by two-dimensional high performance liquid chromatography.
Current developments in drug discovery in the pharmaceutical industry require highly efficient analytical systems for protein mapping providing high resolution, robustness, sensitivity, reproducibility and a high throughput of samples. The potential of two-dimensional (2D) HPLC as a complementary method to 2D-gel electrophoresis is investigated, especially in view of speed and repeatability. The method will be applied for proteins of a molecular mass <20 000 which are not well resolved in 2D-gel electrophoresis. The 2D-HPLC system described in this work consisted of anion- or cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. We …
Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties
The determination of proteins utilising a polymer-based restricted access suppor material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a mincrobore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor.
Is multidimensional high performance liquid chromatography (HPLC) an alternative in protein analysis to 2D gel electrophoresis?
The interactive modes of High Performance Liquid Chromatography (HPLC) of proteins provide a platform for the construction of a multidimensional HPLC system coupled to mass spectrometry. We present a system composed of both anion and cation exchanger columns, in the first dimension, and n-octadecyl bonded 1.5 μm nonporous silica columns in the second dimension. Both columns are operated under gradient conditions. A system suitability test with standard proteins showed that the total analysis can be performed within about 20 minutes. The fractions taken from the ion exchanger column are directly analyzed within one minute on the reversed phase column at a high flow rate. Two reversed phase c…