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We perform the analysis using reverse phase chromatography coupled directly to high resolution mass spectrometer. Next, we use the MS-MS spectra for Mascot database searching. Typically, we search the data against in-house protein databases downloaded from UniProt. The Uniprot database contains all known non-redundant protein sequences or custom designed databases.
The Mascot software finds matching peptides in the database by their peptide masses and peptide fragment masses. It bases peptide identification on a probability-scoring algorithm (www.matrixscience.com). Thus, the results show the significant best matching peptide.
You will find the identified database peptide sequences in the results together with the obtained mass spectrometric peptide maps. The sequence highlights the peptide (s) used for the identification. A list of the matching peptides makes it easy to compare the determined and calculated values.
Lyophilize or speed vac the peptide to a solid sample to increase peptide stability during shipment.
Alternatively, freeze the sample or refrigerate to +4 oC for cold shipment of the liquid sample.
Avoid detergents, DMSO, glycerol and other non-volatile solvents. Keep buffer concentration at a minimum; high salt amounts interfere with the ionization in the MS. LC-ESI MS is sometimes possible for samples containing small amounts of salts or urea. However, the best results are obtained with low buffer strength in volatile solvents without detergents.
Use Eppendorf Safe Lock tubes or similar high-quality tubes from your lab.
OBS. If your sample contains any type of detergent, you should send us a request through the contact form to hear about the analytical capabilities for your project.
