title: Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis.
abstract: Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations.
authors.affiliation: Institut für Polymerforschung Dresden e.V. Abteilung Biokompatible Materialien, Hohe Str. 6, 01069 Dresden, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Salchert
authors.forename: Tilo
authors.initials: T
authors.lastname: Pompe
authors.forename: Claudia
authors.initials: C
authors.lastname: Sperling
authors.forename: Carsten
authors.initials: C
authors.lastname: Werner
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D011506:Proteins