Protein Sequencing - Mass Spectrometry

Mass Spectrometry

The other major direct method by which the sequence of a protein can be determined is mass spectrometry. This method has been gaining popularity in recent years as new techniques and increasing computing power have facilitated it. Mass spectrometry can, in principle, sequence any size of protein, but the problem becomes computationally more difficult as the size increases. Peptides are also easier to prepare for mass spectrometry than whole proteins, because they are more soluble. One method of delivering the peptides to the spectrometer is electrospray ionization, for which John Bennett Fenn won the Nobel Prize in Chemistry in 2002. The protein is digested by an endoprotease, and the resulting solution is passed through a high pressure liquid chromatography column. At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high positive potential into the mass spectrometer. The charge on the droplets causes them to fragment until only single ions remain. The peptides are then fragmented and the mass-to-charge ratios of the fragments measured. (It is possible to detect which peaks correspond to multiply charged fragments, because these will have auxiliary peaks corresponding to other isotopes - the distance between these other peaks is inversely proportional to the charge on the fragment). The mass spectrum is analysed by computer and often compared against a database of previously sequenced proteins in order to determine the sequences of the fragments. This process is then repeated with a different digestion enzyme, and the overlaps in the sequences are used to construct a sequence for the protein.