Recovery of gel-separated proteins for in-solution digestion and mass spectrometry

A protocol for mass spectrometry of gel-separated proteins resulting in sig nificantly increased sequence coverage and in improved possibilities for de tection and identification of posttranslational modifications was developed . In relation to the standard in-gel digestion procedure, the sequence cove rage using a combination of matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry was on the average increased by 30%. The method involves electroblotting of the gel-separated proteins to a poly(vinylidene difluoride) membrane. The proteins are extracted from the membrane using a solution of 1% trifluoroacetic acid in 70% acetonitrile an d lyophilized. After reconstitution of the protein extract in digestion buf fer, proteolytic cleavage is carried out in-solution as opposed to the stan dard in-gel digestion procedure. This allows recovery of large and hydropho bic peptides for mass spectrometry and reduces the risk for entrapment of p roteolytic peptides in the gel matrix. The method was applied to proteins i n the 30-40-kDa range with highly different structural properties. The impr oved ability to localize and determine protein modifications is shown for N -terminal acetylation and methylation of a histidine residue. Furthermore, the method enables fast screening of homologous protein sequences.