hEGF peptide mapping

[Astindoram]

Hi everyone, I´m on a Master program and next week I have to go to a Biotec company to solve a problem in just 1 week... I´ve been asked to characterise the human epidermal growth factor at least with two proteases. This protein is very small around 6kDa, and I´ve never done a peptide map of any protein. If anyone could guide me through the basics would be great. I need to understand what should I know about the protein and how to select the appropriate proteases, and how to actually see in a simple experiment the peptide map, like in electrophoresis.

Thank you.

 

Alvaro

Edited July 28, 2014 by Astindoram

[iRNAblogger]

So I don't have any direct experience with this topic, but from my reading, I would start with a trypsin digest of the protein. You can look at this paper to see the basics: http://www.ncbi.nlm.nih.gov/pubmed/3930949

 

Another method is using cyanogen bromide. If your protein has any methionines in it, then you can get relatively large protein fragments using this chemical for cleavage.

 

The two ways to characterize the peptide fragments would be Edman Degradation or Mass Spec. Or if all you are looking for is simple information such as "how many methionines are in this protein" then you can use cyanogen bromide and simple SDS-PAGE to figure that out. If you are curious how many lysines or arginines, a similar experiment using trypsin and SDS-PAGE would work. But it depends on what kind of information you are looking for.