Enzymatic protein digestion
Before being analyzed by w88 slot 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/w88 slot (LC/MS)
- Thermo Scientific Orbitrap w88 slot with ETD, coupled to a Thermo Ultimate 3000 nanoLC system.
- Bruker w88 slot Pro2, coupled to a Bruker NanoElute LC.
- Evosep One LC is primarily used for clinical samples such as whole blood, serum, and urine. Can be coupled with w88 slot Eclipse or TimsTOF depending on experiment
- Protein Identification can be performed on w88 slot gel bands or solution.
- Proteome characterization is w88 slot label free or isobarically labeled approaches.
- Tandem Mass Tags (TMT) are analyzed w88 slot multi-dimensional HPLC. The first dimension is basic RPHPLC followed by traditional reversed phase HPLC on the Eclipse.
- Data-Independent Acquisition (DIA) is performed using w88 slot Eclipse or the timsTOF and the data processed using Biognosy's Spectronaut software.
- Targeted proteomics is performedon w88 slot Eclipse or timsTOF.
- Data analysis is w88 slot Biognosys Spectronaut or Thermo Scientific’s SEQUEST® and Proteome Discoverer. Data output is provided to the user through either Spectronaut or Proteome software Scaffold with both having free viewers.
Requirements
50 fmol of tryptic digest on column; w88 slot in an aqueous solution of <5% acetonitrile, <0.01% (w/v) detergent concentration. Contact the Center to discuss needs or questions.
