Identification of proteolytic degradation of recombinant protein

Alphalyse
May 9. 2015

Question:

I purified a recombinant protein and analyzed it by 1D SDS PAGE. In addition to the intact protein (52 kDa), there are 3 more bands presumably from proteolytic degradation of the protein. I would like to identify the 3 bands, with the aim to find out where in the sequence the proteolytic cleavage takes place. What analysis will you recommend and can the analysis be performed on the gel bands or protein in solution?

 

Answer:

The additional protein bands in the gel could be proteolytic degradation products. Alternatively, they may be co-purified contaminating proteins from the host cell and expression system. To quickly find out, simply cut out the gel bands and send them to Alphalyse for the Protein identification service. We will digest the gel band proteins with trypsin, do MALDI MS/MS analysis on the peptide mixture, and finally perform a database search for protein identification. The database contains all known protein sequences, including all common host cell proteins.

To characterize proteolytic cleavage in degradation products in more details, requires larger protein amounts  in-solution

First we will purify the protein forms by liquid chromatography (rp-HPLC). Then we perform 2 analyses:

1. Mass spectrometric peptide mapping. The HPLC fractions are digested with a protease (typically trypsin) and the peptides analyzed by nano LC MS/MS. We correlate the peptides and MS/MS data to the exact amino acid sequence of your recombinant protein. The sequence coverage of the peptide map thus shows the protein part that is present in each degradation fragment.

2. Top-down protein sequencing by MALDI ISD of each HPLC fraction. This analysis fragments the protein from both termini. Comparing with the full length protein sequence then determines the exact site of proteolytic cleavage. The MALDI ISD analysis typically confirms 20-70 amino acid residues from both C-terminal and N-terminal.

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

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