Complex Protein Mixture Analysis:

A complicated mixture of proteins can be analyzed via LC/ESI/MS/MS however not all the proteins in the mixture may be identified. The technique depends on the intensities of peptide ions as they elute off of a reverse phase HPLC column (100 micron ID) during a 1 hour gradient. When an ion reaches a predetermined threshold it triggers an MS/MS acquisition. This process is repeated during the course of the HPLC separation so that at the end of the run one may obtain 1000 MS/MS spectra which can be correlated to the databases to identify the original protein components. Even though a number of ions can be analyzed in a single chromatographic peak the technique is still time dependent and subject to ion suppression effects. What this means is that in a digest mixture of 30 proteins you may only observe the 10-20 most abundant proteins during a single HPLC run. The number of proteins identified is strongly dependent on the relative abundances of the individual components. One way of getting more complete coverage of a complicated mixture of proteins is to first resolve them by one dimensional SDS PAGE. Once resolved the gel is sectioned into 10-30 slices down the entire lane. Each slice is then analyzed after in gel trypsin digestion by a LC/ESI/MS/MS run. This method sometimes referred to as GeLC can become quite costly depending on the number of gel slices analyzed. An example study where this technique was used in my lab can be found at (Pazour et al. J. Cell Biol. 2005 170: 103-113).