Protein identification from gel bands / spots
Protein bands/spots of interest are excised from the gel and digested in-gel with a site specific protease such as trypsin. The tryptic peptides are eluted from the gel matrix and subjected to mass spectrometric analysis. Single protein bands/spots are usually analysed by MALDI tandem Time-of-Flight mass spectrometry. Alternatively LC-coupled tandem mass spectrometry can be performed to gain higher sequence coverage and to identify post translational protein modifications as well as other co-migrating proteins of lower abundance. For low complexity samples we usually perform nano-flow RP-LC coupled LTQ-Orbitrap mass spectrometry with a very short LC-gradient.
Proteins are then identified by matching the measured collision induced dissociation (CID) pattern of tryptic peptides with the calculated fragmentation pattern of tryptic peptides predicted from in silico digested sequence databases. We usually use Mascot and SEQUEST search engines to interrogate various sequence databases. Either amino acid or nucleotide sequences can be used.
The goal of shotgun proteomics is the identification of all proteins in a certain protein complement including their post-translational protein modifications and the acquisition of quantitative information. 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 a hydrolytic endopeptidase. The resulting peptides are extracted from the gel matrix and further fractionated by liquid chromatography. The liquid chromatography is usually inline-coupled to nanospray ionization tandem mass spectrometry. Off-line coupling to MALDI-TOF/TOF MS is also possible.
In a gel-free approach the protein extracts are purified, buffer exchanged and digested in-solution using procedures such as the filter-aided sample preparation or S-Trap purification. The resulting peptides can be further pre-fractionated by off-gel isoelectric focusing or high pH reversed phase chromatography prior to LC-coupled tandem mass spectrometry.