Introduction
The protein samples will be reduced and alkylated with iodoacetamide, i.e. carbamidomethylated, and subsequently digested with Trypsin that cleaves after Lysine and Arginine residues. The resulting peptides are concentrated by Speed Vac lyophilization and redissolved for injection on a Dionex nano-LC system and MS-MS analysis on a Bruker Maxis Impact Q-TOF instrument. The MS-MS spectra are used for Mascot database searching. The data are searched against in-house protein database downloaded from UniProt containing all known non-redundant protein sequences.
Database search
The Mascot software finds matching proteins in the database by their peptide masses and peptide fragment masses. The protein identification is based on a probability-scoring algorithm (www.matrixscience.com) and the significant best matching protein is shown in the Results. Homologous proteins with a lower score are not included in the report. If the protein from the correct organism is not present in the database, then a significant matching homologous protein from another organism is reported. If several proteins are identified with a significant score then several protein identifications are reported for the sample.
Reporting
The identified database protein sequences are shown in the Results together with the obtained mass spectrometric peptide maps. The peptides used for the identification are highlighted in the sequence and the matching peptides are listed for comparison of the determined and calculated values.The same peptide can appear as multiple identifications. It is considered a positive identification when at least 2 peptides have an Ions score above 20 or if a protein under 20 kDa has 1 peptide with an Ions score above 50. The sequence coverage is not considered for the identification. The total Mascot score provided for each identification is a total of all the individual peptide Mascot scores.
Sample preparation
In-solution samples
Protein samples can be submitted in solution or lyophilized. Amount ~ 5-50 micrograms
Avoid detergents, DMSO, glycerol and other non-volatile solvents. Keep buffer concentration at a minimum as high salt will interfere with the ionization in the MS. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents.
Use a Eppendorf Safe Lock tubes, or similar tubes from your lab
Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
Alternatively, freeze the sample or refrigerate to +4 oC for cold shipment of the liquid sample.
