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)
- Thermo Scientific Orbitrap Eclipse with ETD, coupled to a Thermo Ultimate 3000 nanoLC system.
- Bruker timsTOF Pro2, coupled to a Bruker NanoElute LC.
- Evosep One LC is primarily used for clinical samples such as whole blood, serum, w88 casino games loginand urine. Can be coupled with either the Eclipse or TimsTOF depending on experiment
- Protein Identification can be performed on either gel bands or solution.
- Proteome characterization is performed using either label free or isobarically labeled approaches.
- Tandem Mass Tags (TMT) are analyzed using 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 either the Eclipse or the timsTOF and the data processed using Biognosy's Spectronaut software.
- Targeted proteomics is performed on either the Eclipse or timsTOF.
- Data analysis is performed using either 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; sample w88 online casinoin an aqueous solution of <5% acetonitrile, <0.01% (w/v) detergent concentration. Contact the Center to discuss needs or questions.