Mass Spectroscopy

Recent advances in protein methods have led to the application of mass spectrometry to the identification of proteins by mass mapping. In this process, targeted proteins are isolated by SDS gel electrophoresis and are digested directly in the gel slice with trypsin or Endo LysC C endoproteinase. The resulting peptides are extracted and the unfractionated mixture is analyzed by MALDI-TOF mass spectrometry. The mass measurements of the resulting peptides are used to query a database of protein and DNA sequences for likely candidate proteins. This is accomplished by matching the measured masses with masses predicted from the databases after 'virtual' digestion with the proteinase. The more peptide masses that match the predicted masses, the more certain one is of the likelihood that the protein is identified. Clearly, high-mass accuracy is required for this method to be of use. Sometimes no matches are found or the level of certainty is too low. Working with species whose genome is complete is a benefit in experiments of this nature.

The ThermoFinnegan DecaXP ion trap mass spectrometer gives us the opportunity to perform limited peptide sequencing to aid in protein identification. The mass spec can be programmed to automatically perform ms/ms to obtain fragmentation and sequencing information and is equipped with Sequest software for automated database searches using peptide mass and fragmentation information to aid in protein identification.

It is possible, also, to combine the complementary techniques of mass spectrometric mapping and chemical protein sequencing (Edman degradation) to identify proteins. This is especially true when you are working in 'non-genome' species. Amounts of material required for the mass mapping approach are dependent upon the sample and its preparation. In our laboratory, with our validated procedures, we are able to perform mass mapping/protein identification experiments with as little as 2 pmol (loaded on the gel) of sample. However, more sample increases the chances of a successful outcome. The success of any experiment will depend upon reasonable sample estimates and very careful sample handling. We recommend that you contact us before planning any mass mapping experiments to help you estimate the amount of protein and describe preferred handling techniques.

If chemical sequencing is called for in the experiment, it is necessary to begin with more sample than is required for mass spectrometric analysis alone. Successful experiments are routinely performed in the range of 20-50 pmol of protein. This relatively large amount of material is necessary due to losses during digestion, extraction and HPLC purification.