Mass Spectrometry Lab
Mass Spectrometry Protocols
The Mass Spectrometry Lab of the Nevada Proteomics Center offers a variety of services. These include enzymatic protein digestion and LC-MS/MS.
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, 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.
LC/MS (Liquid Chromatography/Mass Spectrometry)
- 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 by manual injection and using a quick LC gradient of 15 minutes.
- Protein Identification from excised 1D PAGE gel plugs is performed by manual injection and using an LC gradient of 60- 90 minutes.
- Protein Identification from complex samples is performed using multi-dimensional LC. The first dimension is either strong cation exchange (SCX) or Hydrophilic Interaction Chromatography (HILIC) followed by reversed phase LC. 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. Alternatively, the first dimension separation may be accomplished by isoelectric focusing on an Offgel fractionator.
- Targeted proteomics are performed using a Thermo Scientific Vantage triple quadrupole mass spectrometer coupled to a Thermo EASY nLC 1000 and a New Objectives PicoChip source.
- Data analysis is performed using Thermo Scientific’s SEQUEST® and Proteome Discoverer. Data output is provided to the user as a Scaffold output file.
Requirements: >50 fmol of tryptic digest on column; sample in an aqueous solution of <5% acetonitrile, <0.01% (w/v) detergent concentration.