Protein ID by nanoLC-MS/MS

  • Confirm the identity of a purified protein
  • Identify unknown proteins in complex samples
  • Detect proteins in pull-down experiments

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

The service include digestion using highly specific protease to generate peptides from t...

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

The service include digestion using highly specific protease to generate peptides from the protein(s). 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 reviewed public Swiss-Prot database. To ensure access to the newest data, we update the databases on a monthly basis .

The nanoLC-MS/MS service includes:

  • You provide us with gel band containing one or multiple proteins.
  • We perform in-gel trypsin digest and then nanoLC-MS/MS peptide analysis.
  • We then search the data against protein sequence database for protein identity.
  • Identification of typically up to 10-40 proteins per sample.
  • Finally, you receive the report within 5-10 working days.

Please send us a description of your project if you need a tailored project proposal. 

MoreLess details

The process:

  • 1 You provide us with your sample
  • 2 Analysis with nanoLC-MS/MS and data search against relevant databases
  • 3 We determine 10-100 proteins per sample
  • 4 You receive the report within 4-8 work days

More information:

See an example of how results are presented

Get sample report Identify multiple proteins by nanoLC-MS/MS

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We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

Protein identification process with nanoLC-MS/MS

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, which contains 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 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 instead report a significant matching homologous protein from another organism. If we identify several proteins with a significant score, then we report several protein identifications for the sample.

Reporting

The Results show the identified database protein sequences together with the obtained mass spectrometric peptide maps. The sequence highlights the peptides used for the identification and lists the matching peptides for comparison of the determined and calculated values.

The same peptide can appear as multiple identifications. Therefore, it is considered a positive identification when at least 2 peptides have an ion score above 20 or if a protein under 20 kDa has 1 peptide with an ion score above 50. The sequence coverage is not considered for the identification. The total Mascot score provided for each identification is thus a total of all the individual peptide Mascot scores.

Sample preparation

Samples can be submitted as in-gel or in-solution

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 normal. 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 normal. After staining with Coomassie, excise selected areas with multiple spots. Place each piece (2x5mm) in separate tubes and ship these for analysis.

NOTE: All gel samples should be cut out as 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, the best results are obtained with low buffer strength in volatile solvents without detergents.

 

OBS. If your sample contains detergents in any sort, send us a request through the contact form to hear about the analytical capabilities.

 

short 1d page long 1d page 2d page for nanoLC-MS/MS analysis

Three different ways for preparing complex in-gel samples for protein identification by nanoLC-MS/MS

Meet the experts

For this type of analysis our experts include:

Do you need help?
Martin Villadsen

MSc in Engineering within Medical Biotechnology

Read more
Do you need help?
Li Peng Lundgren

PhD in Protein Chemistry

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