Instruments & Methods

 

A description of the protein identification service

Protein Bands and spots will be excised once the gel is received. And they will be stored in 4 ºC refrigerator until they are analyzed. In-gel digestion is performed by ms-compatible in-gel digestion protocols. All efforts will be devoted to prevent any contamination during this step. Modified porcine trypsin is used in digestion and typical time will be overnight. After in-gel digestion, the peptide mixture is dissolved and subjected to nano LC/MS/MS analysis. Nano-HPLC / electrospray mass spectrometry is performed on a ThermoFinnigan LTQ instrument, coupled with an Agilent 1100 Series HPLC system. The separation is achieved with a 75-micron i.d. C18 reverse-phase chromatography column. The resulting files are searched against NCBI-nr protein sequence databases through the Mascot search engine (Matrix Science.). Protein identification could also be limited due to the lack of complete database information for the specific species. In that case the files will be searched against a broader category and redundant sequences might be used to provide protein identification. All confident results from database searching will be provided in the result. Keratin-like proteins will also be provided as long as it can be positively identified in our analysis.

Instruments for the facility service
ThermoFinnigan LTQ MS: featuring nanospray ionization source, Xcalibur 1.4 SR1 software

Agilent 1100 HPLC systems: featuring Capillary or Nano Pumps, auto sampler and manual injector.