Workflows » High throughput protein identification/proteomics
The goal of high throughput proteomics is basically the identification of all proteins in a certain protein complement including their post-translational protein modifications and quantitative information (see below). It can be performed either as a gel-based or a gel-free approach.
The gel-based approach includes the pre-fractionation and purification of proteins by 1-dimensional protein gel electrophoresis. The gel is then fractionated into several molecular weight fractions to reduce sample complexity and proteins are in-gel digested with trypsin. The tryptic peptides are extracted from the gel matrix and further fractionated by 1- or 2-D liquid chromatography. The liquid chromatography is coupled either off-line to MALDI-based or in-line to ESI-based mass spectrometry.
In a gel-free approach the sample complexity should be reduced on the protein level by various fractionation strategies such as a serial protein extraction followed by liquid chromatography. The proteins are then digested in-solution and the proteolytic fragments further fractionated by 1-D RP-LC or 2-D chromatography using SCX (strong cation exchange chromatography) in combination with RP (reversed phase)-LC. The liquid chromatography is coupled either off-line to MALDI-based or in-line to ESI-based mass spectrometry.