Proteinase K instrument settings?

[Toxic]

Hi all. We are just about to do some Proteinase K cell membrane shaving experiments on some Mycoplasma because some of the proteins we are interested in have sequence stretches of a few hundred amino acids with no K or R. But I'm wondering what instrument settings people use when analysing proteinase K digests, mainly whether they use data dependant acquistion or just try and fragment everything. It's definite that a number of peptides in this sample will be singly charged due to the lack of K and R, but I've seen papers where DDA methodologies are used. It's one of those things that seem to be ignored in people's method sections.

Any thoughts or advice would be greatly appreciated. The sample will be going through my Tempo/QSTAR Elite nanospray system, although I am thinking of trying to fractionate it and analyse on our 5800 MALDI system (although I don't have a spotter). Cheers.

[Toxic]

Anybody? Someone must have some thoughts or opinions on this?

[Doug]

I can't advise you on instrument settings. But I have seen papers describing chemical derivitization of peptides in order to increase charge state. If there is one protein you really need to see you might consider it.

[url=Krusemark CJ, Frey BL, Smith LM, Belshaw PJ. Complete chemical modification of
amine and acid functional groups of peptides and small proteins. Methods Mol
Biol. 2011;753:77-91. PubMed PMID: 21604117.]Krusemark CJ, Frey BL, Smith LM, Belshaw PJ. Complete chemical modification of
amine and acid functional groups of peptides and small proteins. Methods Mol
Biol. 2011;753:77-91. PubMed PMID: 21604117.[/URL]

[Artur]

Why are you using a nonspecific protease?

[Toxic]

We're using proteinase K because it's what we have in the fridge and the sequence doesn't really lend itself to other enzymes cutting it, aside from acid hydrolysis which will be next on the list if this doesn't work.

[Toxic]

But I have seen papers describing chemical derivitization of peptides in order to increase charge state. If there is one protein you really need to see you might consider it.

Awesome, Doug. That's extremely useful for something else I need to do. Cheers.