MS/MS ion search method

The MS/MS ion search method is very effective in identifying multiple proteins that coexist even after SDS-PAGE or two-dimensional electrophoresis following pull-down procedures.

Multiple proteins are often included in a gel section even after being processed by a method with a high protein-separation capacity, such as two-dimensional electrophoresis. To analyze complex constituent proteins that noncovalently bind to specific proteins, protein complexes obtained by pull-down procedures are separated by SDS-PAGE. Here again, bands include multiple proteins. The MS/MS ion search method is extremely effective in identifying such multiple proteins. For the MS/MS method, a tandem-type mass spectrometer is used. In the tandem-type mass spectrometer, two mass spectrometers are connected in tandem. Between the two mass spectrometers is an ion selector for selecting only a specific ion. In TOFTOF mass spectrometers, ions are separated in the first TOF stage. Just before a specific ion reaches the ion selector, the selector voltage is turned off. Then, after the ion of interest passes the ion selector, its voltage is turned on to allow only the ion of interest to pass. The ion of interest is then disintegrated by collision with inert gas in a collision cell and accelerated again to be separated at the second TOF stage. During disintegration in the collision cell, mainly the framework structures of peptides are broken at random. The MS/MS spectrum represents information about the amino acid sequence of the peptide of interest.
For example, if it is assumed that leucine can be distinguished from isoleucine and lysine can be distinguished from glutamine when five consecutive residues of the amino acid sequence can be determined, one amino acid sequence can be narrowed down out of 205 amino acid sequences. Since partial sequences of multiple peptides can often be analyzed for a specific protein, the protein can be identified with high precision.
The procedures from post-electrophoresis gel treatment to desalting are the same as those explained for the PMF method.

1. Acquire mass data of MS/MS fragments of individual peptides by MS/MS analysis.
2. Search a protein database for theoretical values of MS/MS fragments of peptides obtained by trypsin digestion of proteins. Compare the theoretical values to the actual measured values, and select those that have a high relevance ratio.

Principle of the MS/MS ion search method