Characterization Of Protein Mixtures Research Articles

Characterization of factor VIII pharmaceutical preparations by means of MudPIT proteomic approach

For a good clinical outcome of Haemophilia A substitutive therapy a detailed characterization of factor VIII (FVIII) concentrates is required, in order to disclose the eventual relations between differently composed concentrates and their biological effects. This preliminary work could be a first step towards a deep structural characterization of FVIII concentrates, using the fast and simply manageable MudPIT technology, which enables the identification and characterization of protein mixtures taking advantage of both the high separation capacity of two-dimensional chromatography and the powerful peptide characterization ability of tandem mass spectrometry. The aim of this study was to evaluate the suitability of for the characterization of FVIII molecule in complex mixtures such its commercial concentrates, both plasma-derived and recombinant, and for the determination of the protein composition of different FVIII preparations. By means of Multidimensional Protein Identification Technology (MudPIT) it was possible to assess the presence of factor VIII in its preparations and to identify most of the contaminant proteins without gel separation. In particular, 125 and 42 proteins were identified in plasma-derived and recombinant concentrates, respectively. Concerning investigation of FVIII, 24 different peptides were identified in plasma-derived corresponding to 7, 29, 27, 19 and 67 of percentage coverage for A1, A2, A3, C1 and C2 domains, respectively. About its multimeric carrier von Willebrand factor (VWF), we have sequenced 42% of domain interacting with A3 and C2 domains of FVIII. Finally, it has been observed that normalized parameters, such as total peptide hits obtained by SEQUEST may be used for evaluation of the relative abundance of FVIII in different preparations.

Characterization of protein mixtures by ion-exchange chromatography coupled on-line to nuclear magnetic resonance spectroscopy

The first example of HPLC–NMR analysis of proteins is reported. By this means, a mixture of hen egg lysozyme and horse heart cytochrome was separated and identified. The chromatographic separation is based on ion-exchange HPLC, which is likewise for the first time coupled to 1 H NMR. In the stop-flow mode, characteristic one-dimensional 1 H spectra, two-dimensional total correlation data sets (TOCSY), and nuclear overhauser effect correlation spectra (NOESY) of the two proteins were obtained. In addition, lysozyme from crude hen egg white was unambiguously characterized by using a stop-flow HPLC–NMR-TOCSY experiment. These experiments extend the applicability of HPLC–NMR to the rapid analysis of protein mixtures.

Packed capillary liquid chromatography/electrospray ionization/mass spectrometry in the pharmaceutical sciences: Characterization of protein mixtures

AbstractThe coupling of packed capillary liquid chromatography with electrospray ionization mass spectrometry has yielded an instrument capable of generating molecular weight information from very complex natural protein mixtures. The use of the packed capillary liquid chromatography columns offers two principle advantages over conventional liquid chromatography columns: high separation efficiencies and very low mobile phase flow rates (100 nL min−1). The low mobile phase flow rates permit the use of a relatively high ionic strength buffer (0.1% trifluoroacetic acid), While delivering a very low mass flux of the buffer to the mass spectrometer.