Enzymatic protein digestion
Before being analyzed by mass spec, most proteins must be broken into smaller fragments. This is accomplished by digestion with a protease, usually trypsin and Lys-C, but may be another proteases of your choice.
Requirements
>100 fmol protein, may be a protein in solution, a gel slice or spot that is visible by coomassie staining.
Liquid chromatography/mass spectrometry (LC/MS)
- LC/MS analysis is performed using a Thermo Scientific Orbitrap Fusion with ETD coupled to a Thermo UltiMate 3000 nanoLC system and a New Objectives digital PicoView source.
- Protein Identification from excised 2D PAGE or DIGE gel plugs is performed using a quick LC gradient of 30 minutes.
- Protein Identification from excised 1D PAGE gel plugs is performed using an LC gradient of 60- 90 minutes.
- Proteome characterization is performed using Tandem Mass Tags and is analyzed using multi-dimensional HPLC. The first dimension is basic RPHPLC followed by traditional reversed phase HPLC. The first dimension is fractionated using a Thermo Scientific UltiMate 3000 LC system. Collected fractions are then analyzed using the Fusion over a two hour gradient.
- Targeted proteomics are also performed using the Thermo Scientific Fusion mass spectrometer.
- Data analysis is performed using Thermo Scientific’s SEQUEST® and Proteome Discoverer. Data output is provided to the user as a Scaffold output file and Ingenuity Pathway Analysis.
Requirements
>50 fmol of tryptic digest on column; sample in an aqueous solution of <5% acetonitrile, <0.01% (w/v) detergent concentration.
