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

Technical details, sample info, experts etc.

NanoLC-MS/MS analysis is the preferred method for identifying proteins – in either 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 highly specific pro...

The service includes digestion using a highly specific protease to generate peptides from the protein(s). We separate peptides using nano-flow HPLC coupled to a High-Resolution mass spectrometer to achieve the highest sensitivity. 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

Services

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

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

$300.00

Results in 2 weeks

Order this service

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

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

$500.00

Results in 2 weeks

Order this service

Order the service here

You can easily order online. If you would like guidance or a quote please contact us.

Order online

This product is available to order online. Enter your sample details and pay online or by PO/invoice

Order online

Contact us by email

Not sure wich to choose? Contact us before you order.

Open contact form

Knowledge center

More information

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

Download and links

Learn more in this protocol and blog posts:

Protocols for gel silver staining

Related blog posts:

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

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 for comparison of the determined and calculated values.

The same peptide can appear as multiple IDs. Therefore, it is considered a positive ID 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 used for identification. The total Mascot score provided for each ID is thus a total of all the individual peptide Mascot scores.

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

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Martin Villadsen

MSc in Engineering within Medical Biotechnology

Explore

Li Peng Lundgren

PhD in Protein Chemistry

Explore