Description of LC MS/MS Analysis
Protein Digests are analyzed using LC MS/MS on either a Waters/Micromass AB QSTAR Elite or a Thermo Scientific LTQ-Orbitrap XL mass spectrometer. Both systems are equipped with Waters nanoACQUITY ultra high pressure liquid chromatographs (UPLC) for peptide separation (1). The instrument choice (determined by the Keck Staff) depends on the project and the level of sample available.
All MS/MS spectra are searched in-house using the Mascot algorithm (Hirosawa et al, 1993) for un-interpreted MS/MS spectra after using the Mascot Distiller program to generate Mascot compatible files (http://www.matrixscience.com/home.html for details). The Mascot Distiller program combines sequential MS/MS scans from profile data that have the same precursor ion. A charge state of +2 and +3 are preferentially located with a signal to noise ratio of 1.2 or greater and a peak list is generated for database searching. Either the NCBInr, a species specific, or a custom database (in FASTA format) are used for searching. All Mascot search results are loaded into the YPED online viewing system for dissemination to the investigator.
- Sample Amount=500 attomols of peptide is routinely identified
- Recommended amount is 1-10 fmol (or > - see figure)
- Coomassie blue and digestion compatible silver stained protein bands are appropriate
- Stone, K.L. and Williams, K.R (2009) Reverse-Phase HPLC Separation of Enzymatic Digests of Proteins in The Protein Protocols Handbook, 3rd edition (Walker, J., ed.) 937-946.