EP3 Fundamentals of Protein Sequence Characterization by Mass Spectrometry.

The first section of the tutorial will describe the instrumentation typically used in biological mass spectrometry applications related to protein identification. We focus on the relevant ionization techniques, common mass analyzers, and sample introduction systems. Attention will be given to properties, such as mass accuracy and mass resolution, which are important to protein characterization and database search strategies for protein identification. Practical considerations regarding the selection and use of instruments as well as troubleshooting information will be offered throughout the presentation. The fundamentals of basic protein sequence characterization, including post-translational modifications, by mass spectrometry will be presented in the second section of the tutorial. Emphasis is placed on the use of tandem mass spectrometry at the peptide level to confirm and in some cases derive partial peptide sequence, identify post-translationally modified sequences, and localize the specific site of attachment. We will describe the basic principles of peptide fragmentation by collision-induced dissociation and how to use these principles to interpret MS/MS spectra. Basic sample preparation protocols compatible with mass spectrometry analysis will be described. The third section of the tutorial will focus on mass spectrometric analyses of protein mixtures (proteomes). Besides shear numbers of proteins, the range of concentrations in certain samples is frequently an impediment to a complete analysis. Various fractionation, capture, and depletion methods will be described for dealing with very complex protein mixtures. Some of these capture methods also provide additional information regarding post-translational modifications. A brief description of database search methods for protein identification will be followed by a more extensive discussion of validating the search results. Finally, brief descriptions of protein quantitation methods will be presented, and their various advantages and disadvantages will be discussed.