You send multiple proteins sample in-solution. The sample is digested in-solution with trypsin and the resulting peptides analyzed by nano LC-MS/MS (30 minute gradient). The MS/MS data is searched against the Swiss-Prot protein sequence database. The database search includes variable oxidation of methionine residues. Optionally we can search the data against the complete UniProtKB database or a custom database (please select your choice below).
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.
Place your samples into microtubes and pack them in a suitable transport box. Remember to print and include the sample submission form.
Customers in the USA Alphalyse, Inc. 299 California Avenue, Ste 200 Palo Alto, CA 94306 USA Att: Thomas Kofoed Tel: (650) 543-3193 |
Customers outside of the USA Alphalyse A/S Roedegaardsvej 209 C DK-5230 Odense M DENMARK Att: Anette Rasmussen Tel: +45 6310 6500 |
You can use our prepaid FedEx Air Waybill to quickly ship samples in packages up to one lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF format) within 24 hours.
Then follow these simple instructions:
If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will often exceed one lb./500g.
Select this shipping option if you plan to fill your transportation box with cooling packs. If you plan to use dry ice, you cannot use this Waybill!
The prepaid FedEx Air Waybill can be used to ship samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF format) within 24 hours.
Then follow these simple instructions:
You can use the World Courier service to ship samples of all types.
If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill, which will be invoiced together with your analysis results.
World Courier service is usually a good option if FedEx does not service your location.
With the prepaid World Courier service, you get:
My name is Anette and I am customer support scientist at Alphalyse. I make sure we take good care of your samples. If you need help, e.g. ordering or shipping samples, send me a message - I'm here to help.