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Set up meetingProtein characterization for optimization of manufacturing processes.
Explore customer caseCombining mass spec analyses for mAb characterization and batch-to-batch comparison
Explore customer caseWe would like to help you as much as possible with your project and therefore provide several kinds of customer support:
The protein samples are first reduced with DTT and alkylated with iodoacetamide. Subsequently, they are typically digested with Trypsin, which cleaves after Lysine and Arginine residues. Next, Speed Vac lyophilization concentrates the resulting peptides.
Then follows a redissolving of the sample for injection on a Dionex nano-LC system and MS-MS analysis on a Bruker Maxis Impact Q-TOF instrument. For Mascot database searching, we use the MS-MS spectra. Finally, we search the data against in-house protein databases downloaded from UniProt.org, which contains all known non-redundant protein sequences.
The Mascot software finds matching proteins in the database by their peptide masses and peptide fragment masses. The protein ID uses a probability-scoring algorithm (www.matrixscience.com) and shows the significant best matching protein in the Results. We do not include homologous proteins with a lower score in the report.
If no protein from the correct organism is present in the database, we report a significant matching homologous protein from another organism. We report several protein IDs for the sample if we identify several proteins with a substantial score.
The Results from nanoLC-MS/MS show the identified database protein sequences together with the obtained mass spectrometric peptide maps. The sequence highlights the peptides used for the identification. It also lists the matching peptides to compare the determined and calculated values.
The same peptide can appear as multiple IDs. Therefore, it is considered a positive ID when at least two peptides have an ion score above 20 or if a protein under 20 kDa has one peptide with an ion score above 50. The total Mascot score provided for each ID is thus a total of all the individual peptide Mascot scores. The sequence coverage is not used for identification.
Use Eppendorf Safe-Lock tubes or similar high-quality tubes from your lab.
In-gel samples
Short 1D PAGE: Load the protein sample onto a 1D PAGE gel and run for only 1 cm (5-8 min at 200 V). After staining with Coomassie, excise the entire stained area as one sample (2x5mm gel piece) and send it for analysis.
Long 1D PAGE: Load the protein sample onto a 1D PAGE gel and run as usual. After staining with Coomassie, excise the entire stained area into 5-10 smaller pieces (2x5mm sized). Place each piece in separate tubes and ship these for analysis.
2D PAGE: Load the protein sample onto a 2D PAGE gel and run as usual. After staining with Coomassie, excise selected areas with multiple spots. Place each piece (2x5mm) in separate tubes and ship these for analysis.
NOTE: Cut all gel samples into 2x5mm pieces.
In-solution samples
Minimum sample 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 may work on samples containing small amounts of salts or urea. However, you obtain the best results with low buffer strength in volatile solvents without detergents.
OBS. If your sample contains detergents of any sort, send us a request through the contact form to hear about the analytical capabilities of nanoLC/MS/MS.
Three different ways to prepare complex in-gel samples for protein identification by nanoLC-MS/MS
