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 good sequence coverage and identify other co-migrating proteins or post translational protein modifications. 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 by an in silico digestion of the interrogated sequence database. We usually use Mascot and SEQUEST search engines to interrogate various sequence databases. Either amino acid or nucleotide sequences can be used