|Proteomics Group, Institute of Functional Genomics, University Regensburg|
Polyacrylamide-gel-electrophoresis (PAGE) is one of the most widely applied techniques for protein separation. Thereby, the proteins migrate within a polyacrylamide matrix driven by the force of an electric field. Various denaturing and non-denaturing methods have been developed for different separation strategies such as isoelectric focussing (separation according to net charge), SDS-PAGE (separation according to size) or BN-PAGE (separation of protein complexes). Both one- and two dimensional systems can be applied granting the possibility to separate thousands of different proteins and protein isoforms. The good compatibility to mass spectrometric methods is a further advantage of these methods.
Fast Protein Liquid Chromatography (FPLC) and High Performance Liquid Chromatography (HPLC) are widely used methods for the separation of proteins and peptides. Various properties of the proteins/peptides may be used for separation with regard to the applied technique such as ion-exchange, reversed-phase, hydrophobic interaction, gel filtration and affinity chromatography.
For Electrospray-Ionization-Tandem-Mass Spectrometry (ESI-MS/MS) protein- or peptide-solutions are sprayed in an electric field. The obtained protein-/peptide-ions are desolvated and analyzed in the QToF by an electric field. All incoming ions can sequentially be isolated, fragmented and the obtained fragment ions are ejected towards the detector.
Online-coupling of nano-reversed-phase-HPLC and tandem-mass-spectrometry facilitates the identification of peptides and proteins from proteolytic digests. Thereby, also proteins from crude mixtures can be identified down to the low-femtomole level.