Protein sequencing and characterization

As primary sequence plays an essential role in structural characterization of proteins, it is important to have tools allowing providing a complete or very high sequence coverage. Generally, this cannot be achieved by traditional trypsin digestion because large parts of proteins are not detected or efficiently sequenced.

We propose here two strategies to overcome this limitation and provide you with very high sequence coverages.

 

MELD digestion of purified protein present in solution    

The protein is digested by an enzymatic cocktail after reduction of disulfide bonds and alkylation of free thiol groups. This can only be performed in solution. The resulting peptides are analyzed by LC-MS/MS.

 

Once data recorded, they are submitted to database search using Mascot search engine in order to identify proteins. Amino acid sequences from the original organism have therefore to be available in public databases like SwissProt, NCBI, etc. or should be provided.

 

Alternatively, if the sequence is unknown, de novo partial sequencing can be achieved using PEAKS 7 software. The software builds ladder of amino acids based on the MS/MS fragments of the peptide and the accurate mass of the peptide. Those tags can be submitted to database search to see if homology is found with existing proteins.

 

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What is delivered?

 

A list of proteins identified with relevant statistical values. For these proteins, you will know which peptides were identified.

Report and results will be sent by email.

 

What is needed for these analyses?

 

At least 50 µg of a purified protein are required. Please note that if the buffer is not compatible with the workflow, an additional purification step will be performed.

 

What is the time to get results?

 

Results are generally available in 15 working days

 

Form

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Top-Down sequencing of purified protein

The protein is directly deposited on a MALDI plate with the appropriate matrix, inducing the fragmentation of the protein during the laser shot. Mass spectrum displays peaks allowing determining parts of the sequence. By this technique, N- and C- termini can be sequenced over maximum 60 AA on both sides. It is therefore more recommended for peptides and small proteins. For top down sequencing of larger proteins, please contact us.

 

What is delivered?

 

A list of sequence tags will be provided. For known sequences, a database search can be performed. In this case, proteins identified with relevant statistical values will be provided.

Report and results will be sent by email.

 

What is needed for these analyses?

 

At least 100 µg of a purified protein are required (minimum concentration of 50 µM). Please note that if the buffer is not compatible with the workflow or the solution is too diluted, an additional purification/concentration step will be performed.

 

What is thetime to get results?

 

Results are generally available in 15 working days

 

Form

Request form

 

PTMs characterization

Most of PTMs and chemical modifications are neither labile nor heterogeneous and can therefore be localized during the database search step. Please provide us information concerning potential PTMs and/or modifications present in your samples.

However, some PTMs are trickier to study and need more complex sample preparation:

- N-Glycosylation

This PTM is one of the most difficult to study because of its natural heterogeneity. We propose you to characterize it at two levels:

- The glycopeptides level: LC-MS/MS data are searched for specific signature of glycopeptides allowing glycans to be localized and partially characterized.

- The glycans level: glycans are released thanks to enzymatic process and are analyzed by mass spectrometry. No information about the localization is obtained. This can be performed on complex mixtures such as serum.

Please contact us for further information and feasibility.

For other PTMs (Disulfide bonds, Phosphorylation, others): please contact us for further information and feasibility.

 

Form

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