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Identify multiple proteins by nanoLC-MS

Identification of protein mixtures by nanoLC-MS/MS

NanoLC-MS/MS analysis is preferable for identification of multiple proteins in a complex mixture. The sample format can be in-solution or in-gel bands or spots from SDS PAGE.

The service include digestion using highly specific protease to generate peptides from the proteins. To achieve the highest sensitivity, we separate peptides using nano-flow HPLC coupled to High Resolution mass spectrometer. As a result, thousands of peptides are identified by its mass and fragmentation pattern, and individual proteins are identified by searching these data against protein databases.

We search the data against the public UniProt database. It contains the amino acid sequence of more than 100 million known proteins. We update the databases on a monthly basis to ensure access to all known proteins.

The service includes:

  • You provide us with gel band containing multiple proteins.
  • We perform in-gel trypsin digest and then nanoLC-MS/MS peptide analysis.
  • We search the data against protein sequence database for protein identity.
  • Identification of typically 10-40 proteins per sample.
  • Finally, you receive the report within 4-8 working days.

Please send us a request with a description of your project, so we can tailor your project proposal. 

Services
Identification of single or multiple proteins

ID of multiple proteins by nano LC-MS/MS (in-gel)

  • Trypsin cleavage and nanoLC-MS/MS peptide analysis
  • Search against protein sequence database
  • ID of up to 10-40 proteins

$300.00

Fast and Easy
Will be delivered in 4-8 days
Info / Order
Request quote
Identification of single or multiple proteins

ID of multiple proteins – nano LC-MS/MS (in-solution)

  • Trypsin cleavage and nanoLC-MS/MS peptide analysis
  • Search against protein sequence database
  • ID of up to 10-100 proteins

$500.00

Fast and Easy
Will be delivered in 4-8 days
Info / Order
Request quote

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.

Short 1D PAGE: The protein sample is loaded onto a 1D PAGE gel and run for only 1 cm (5-8 min at 200 V). After staining with Coomassie, the entire stained area is excised as one sample and sent for analysis.

Long 1D PAGE: The protein sample is loaded onto a 1D PAGE gel and run as normal. After staining with Coomassie, the entire stained area is excised into 5-10 smaller pieces. Place each piece in separate tubes and ship these for analysis.

2D PAGE: The protein sample is loaded onto a 2D PAGE gel and run as normal. After staining with Coomassie, selected areas with multiple spots are excised. Place each piece in separate tubes and ship these for analysis.

Avoid detergents and keep buffer concentration at a minimum. 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

shipping
Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box. Remember to print and include the sample submission form.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
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 USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220 Odense SO
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
shipping
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 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:

  • Place your samples into micro tubes. Pack in suitable transport box. Remember to print and include the sample submission form.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

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 most often exceed 1 lb./500g.

shipping
Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of 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:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Remember to print and include the sample submission form.
  • Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

shipping
Get a quote for shipment by World Courier

The World Courier service can be used for shipment of 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. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork
Order analysis now
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