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 the preferred method for identifying proteins in a simple or complex mixture. The sample format can be in-solution, in-gel bands, or spots from SDS gel electrophoresis.

The service includes digestion using a particular protease to genera...

NanoLC-MS/MS analysis is the preferred method for identifying proteins in a simple or complex mixture. The sample format can be in-solution, in-gel bands, or spots from SDS gel electrophoresis.

The service includes digestion using a particular protease to generate peptides from the protein(s). To achieve the highest sensitivity, we separate peptides using nano-flow HPLC coupled to a High-Resolution mass spectrometer. As a result, the method identifies thousands of peptides by their mass and fragmentation pattern.

We then search the data against the reviewed public Swiss-Prot database. To ensure access to the newest data, we update the databases monthly.

The nanoLC-MS/MS service includes:

  • You provide us with a 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 the protein sequence database for protein identity.
  • ID of typically up to 10-40 proteins per sample.
  • Finally, you receive the report within 10-14 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 10-14 days

More information:

See an example of how results are presented

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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 ID process using 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.org, 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 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.

Reporting

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.

Sample preparation

For nanoLC-MS/MS 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 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.

 

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

Three different ways to prepare 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?
Line Kofoed

PhD in Health and Medical Sciences

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Do you need help?
Tine Nielsen

MSc in Biomedicine

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