Proteins are denatured, reduced and alkylated before digestion with trypsin overnight. Samples submitted as gel slices are destained using appropriate protocols. The peptides are analyzed by nano-flow LC-tandem mass spectrometry in data-dependent scanning mode using an ion trap mass spectrometer. The MS/MS data is searched against the predicted fragments ions from the theoretical digests of proteins contained in a comprehensive database such as NCBInr or Swissprot using a proteomics search engine like Mascot (Matrix Science Limited). Peptide mass fingerprints of digested samples using MALDI-TOF-MS can be acquired on selected samples as needed. The analyzed results, containing proteins identified at the 95% confidence level, are provided to our users using our on-line “paperless” system.
The Center for Mass Spectrometry has several mass spectrometers that can be used for proteomics including the Agilent MSD ion trap SL, Thermo Finnigan LTQ, Thermo Finnigan Deca, Applied Biosystems Voyager-STR-DE MALDI-TOF MS, Micromass M@LDI, and Micromass Q-TOF. A selection of helpful protocols that have been successfully used in our laboratory are provided below:
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