Protease-resistant core?

[GRG]

Hello there,

 

I wonder if anyone might be able to explain to me what a protease-resistant core of a protein is, specifically in regard to its amino acid sequence (does it lack protease specific amino acids, e.g. K and R in the case of trypsin?) and means of formation?

 

Many thanks

[CharonY]

Depends on the context. However, often it is used when referring to proteins with a hydrophobic core. In aqueous solutions they are tend to be tightly packed with minimal exposure to the buffer and are therefore not easily accessible.

Edited December 4, 2016 by CharonY

[Elite Engineer]

therefore not easily accessible.

How would one reach the inner core? Chaotropic agent?

[CharonY]

For example, or using other aggressive denaturation approaches.

[BabcockHall]

Surface-exposed loops, by contrast, are sometimes more accessible to proteases.

[CharonY]

Sure, that is why you can get decent fragments out of membrane proteins. The membrane integral parts are annoying, though.

[GRG]

Would it be possible to fragment this protein into peptides by a tryptic in-gel digestion?

[CharonY]

You mean whether the SDS-treatment is sufficient to denature it? Potentially. Typically samples are cooked and dentured via SDS and some resistant proteins may be denatured sufficiently. However, other than for separation (and potentially purification) purposes there is no reason to run a gel. You can as easily treat with SDS and heat. Though you may have to reduce the SDS prior to digest a bit.

[GRG]

Would the protease-resistant core be digestible by trypsin if I wanted to digest the protein into peptides by the means of a tryptic in-gel digest ahead of sending it for analysis by mass spectrometry?

[CharonY]

As I said, why would a gel be needed if it is only one protein? Or to clarify, are you aware which steps during an SDS-gel can potentially lead to denaturation?

 

Also, what do you need to have analyzed for? For identification simple PMF could be enough, assuming you got enough hydrophilic loops. Or do you want to have complete coverage? Are you looking for PTM, and if so where do you suspect them? Or do you need a quantitative analysis? Is this pure protein, a purification, or crude extract?

Edited December 6, 2016 by CharonY