Mass spectrometry
Mass spectrometry in proteomics
In the past decade there has been a revolution in the application of mass spectrometry to the analysis of proteins and peptides. This has been facilitated in part by two ionization techniques, which are capable of producing charged gaseous ions of both peptides and proteins. Electrospray ionization (ESI) involves spraying a liquid solution of the peptide through a fine capillary held at a high potential. This produces charged droplets that are then rapidly desolvated producing charged ions of the peptide, which are then usually directed into a quadrupole type mass analyzer. The other ionization technique, matrix-assisted laser desorption ionization (MALDI) involves co-crystallizing the sample with an organic matrix which strongly absorbs UV laser light. Upon irradiation under vacuum there is an energy transfer from matrix to peptide analyte, which produces gaseous ions that are typically measured by a time-of-flight (TOF) mass analyzer.
To understand many cellular processes it is necessary to rapidly identify components of an organism's proteome (expressed gene products). As the sequencing of the genomes of many model organisms comes to completion the likelihood that a protein from the organism's proteome can be found in the protein database has increased exponentially. This has precipitated a revolution in the application of mass spectrometric techniques in the identification of proteins in the databases (proteomic mass spectrometry). The identification approach involves digesting as little as 10 femtomoles of gel purified protein with a site-specific protease (i.e. trypsin) and then mass analyzing the resultant peptides with mass accuracies about 50 ppm. The peptide masses can then be submitted to one of several mass database search algorithms that are available on web based servers or as user purchased software. Most of these search algorithms generate theoretical mass profiles for each protein in a specified database and compares it with the experimental data to arrive at a statistical correlation. The searches can be based on either the collective mass data from a number of peptides (MS) or the mass of a single peptide and fragments derived from it by a collisional or decay process (MS/MS).
The advantage of searching with MS/MS data is that it is based on the sequence of the peptide and therefore will be more discriminating in any database correlation analysis. Also MS/MS data allows one to analyze protein mixtures more readily since each peptide from a digest will be represented by a single protein component. In addition MS/MS data can be used to search the Expressed Sequence Tag (EST) databases. This is particularly useful since several proteins have typically only a small portion of their sequence represented by EST clones and therefore are not readily identifiable by the MS data from several peptides.
The laboratory is equipped with the Voyager-DE PRO MALDI-TOF (Applied Biosystem) instrumentation, which is currently being used to identify proteins by MS data from peptide mixtures. The laboratory also has a QSTARŪ XL (Applied Biosystem) that is directly coupled to a capillary HPLC system.