Recent advances in protein methods have led to the application of mass spectrometry to the identification of proteins by Peptide Mass Fingerprinting (PMF). In this process, target proteins are isolated by SDS gel electrophoresis and are digested directly in the gel slice with trypsin, other proteases or cleaving chemicals. The resulting peptides are extracted and the unfractionated mixture is analyzed by MALDI-TOF mass spectrometry. The masses 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 or chemical cleavage agent. 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.
Amounts of material required for the mass fingerprinting 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.
Several drawbacks to PMF need to be remembered when planning your experiment. One is the inability to specifically isolate the amino terminus of the protein. Therefore, amino acid sequence information about this region of the molecule is often difficult to obtain using mass spectrometry. Second, due to mass similarities of amino acids it is often difficult to correctly identify amino acids (especially during MS/MS or de novo experiments. For example, leucine and isoleucine have isobaric masses and cannot be differentiated by PMFor MS/MS. Other amino acids (for example, Glutamine and Lysine) have relatively small mass differences and can be mis-identified by these methods.
Correct identification of a recombinant protein (and sequence) can often be accomplished using mass spec and PMF alone, but sometimes direct sequencing by Edman techniques is required. Therefore, it is our preferred approach 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. We highly recommend that you consult with the PCL staff to help you determine the best course of action for your project needs.