Protein Identification and Characterization
Protein identification is a central mass spectrometry-based service offered by the Proteomics Core. This service is most frequently, but not exclusively, performed on stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel bands or spots.
Protein characterization requires the identification and location of post-translational modifications, detection of mutations or truncations, and intact mass molecular weight determination. Requests for protein characterization should not be considered routine, but rather more experimental because the desired outcome is sample-dependent and there is a need for additional personnel effort, instrument time and interactive data analysis.
Before submitting samples, all first-time protein identification and characterization clients must contact the Proteomics Core. The details required for successful analysis are sample dependent and heavily dependent on proper sample handling, so interested users must clearly understand these requirements before submitting samples.
Protein identification. The first step in protein identification is digestion of the SDS-PAGE spot or band with an endoproteinase, such as trypsin. Next, the digested sample is analyzed using nano high-pressure liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) methods.
The Proteomics Core utilizes Thermo Scientific LTQ Orbitraps — coupled with Eksigent NanoLC-2D pumps — for data acquisition, and uses Proteome Software's Scaffold analysis tool to compile the outputs from multiple search algorithms such as Mascot, Sequest and X! Tandem.
Protein characterization. The general strategy for identifying post-translational modifications begins in a similar way as for protein identification: digest the protein with the appropriate enzyme; analyze by nanoLC-ESI-MS/MS; and perform protein sequence searching, allowing for the mass changes of the desired modifications.
The major difference between protein characterization and protein identification is that protein characterization requires manual review and validation of the search results that indicate a modified amino acid.
Detection and location of post-translational modifications is seldom accomplished with a single digest and mass spectrometry run, especially in cases where there are no known sites and a more global interrogation of the protein is the goal. Multiple runs with different enzymes are used to maximize the protein coverage. If specific sites are suspected of being modified, a more focused approach can be used.
Protein identification costs are on a per-band or per-spot basis. Protein characterization includes the protein identification costs, along with additional costs for data and sample analysis using alternative and supportive techniques.
For more information or to initiate a service or project request, email the Proteomics Core.