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Protocol for in-solution digestion (UREA & Acetonitril)
Posted: Mon Oct 13, 2014 12:10 am
I am looking for a standard, but optimized, protocol for in-solution digestion. For example, is it recommendable/important to to add Urea and Acetonitril (50%) to improve the digestion?
A link to a optimized protocol would be great. Thank you.
Posted: Mon Oct 13, 2014 10:38 pm
Previously i used combination of 4M urea and 30% ACN in my protocol but somehow was not happy with the digestion (Based on raw meat analysis and TIC, TCEP and MMTS protocol). Recently started with 8M urea based in solution digestion protocol (DTT and IAA protocol) which is working very well for me.
Not sure about the ACN interference in trypsin activity
m/z wrote:I am looking for a standard, but optimized, protocol for in-solution digestion. For example, is it recommendable/important to to add Urea and Acetonitril (50%) to improve the digestion?
A link to a optimized protocol would be great. Thank you.
Posted: Mon Oct 13, 2014 11:32 pm
Posted: Tue Oct 14, 2014 1:05 am
I would actually recommend switching from Urea to Guanidinium chloride, mostly because it creates less chemical artifacts. Urea can degrade to iisocyanic acid and cause carbamylation of peptides at elevated temperatures (ie. temperatures needed for trypsin lys-c digestion). Another advantage with GndHCl is you can boil the sample without creating any side reaction on peptides, and Lys-C is active in it up to 6M concentrations. I would recommend digesting with Lys-C first at 6M then diluting to under 1M before adding trypsin. Regarding acetonitrile, I would avoid adding it if you're going to desalt your sample (which you should def do if you're running on MS, and separating on C18 column) on a C18 Sep-pak, because you will end up losing all your peptides. I would actually recommend adding triflouroethanol at like 5% instead of acetonitrile, there have been some published reports of it increasing the number of peptides detected when used as an additive. Of course I don't think you will see much of an improvement since the peptides will already be in denaturing conditions when combined with GndHCl or Urea. Feel free to ask if you have more questions.
Posted: Tue Oct 14, 2014 3:55 am
Great stuff Thanks. I will try GndHCl also, and completely avoid ACN.
1) With double digestion do you simply set enzyme as "trypsin" even though Lys-C was also used?
2) Would you advice to use zip tipping with SCX tips in case there are contaminating detergents? Detergents cannot be removed by C18 purification, right? What I always like about Gel-LCMS is that detergents are rarely a problem, since they are removed during electrophoresis...
Posted: Tue Oct 14, 2014 4:13 am
What is exact lysis buffer composition? I mean it is 8M GndHCl in ammonium bicarbonate or TRIS and what is the final pH should be? Do you use DTT/IAA or something else (TCEP, CAA, etc.), at what conc. and temperature? Probably I can find all this info from the literature but I prefer to get it from person who is happy with the protocol.
Posted: Tue Oct 14, 2014 4:59 am
A few more questions:
3) After in-solution digestion the samples are often reported to be treated by "lyophilization". Is this the same as speedvac? Is it best to dry the sample (including Urea/Gnd-HCl) to completion and then dissolve the peptides in 0.1% TFA, or is it better to simply add TFA to the sample?
4) Sep-pak versus zip tip?
You mention Sep-pak, and not zip tipping? Sep-pak can be used with bigger volumes right? So you can perhaps avoid the speedvac step and save time and thus reduce peptide binding to the tube walls?
Posted: Tue Oct 14, 2014 5:09 am
@ m/z - Yes trypsin digestion followed by lys-c. However i'm not sure about the gain in identification when using in combination. At-least the combination will be beneficial when looking for PTMs. About detergent removal once i used pierce removal detergent cartridges, but it didn't work well for me. After in solution digestion,directly i did desalting with Sep-pak cartridges followed by drying them in speed vac. I would recommend don't use TFA, in past, i had lot of problem of ion suppression.
@ Infinity - I used 8M urea in 50 mM ammonium bicarbonate followed by treatment with DTT/IAA
Posted: Tue Oct 14, 2014 5:44 am
Biomarker wrote:@ m/z - Yes trypsin digestion followed by lys-c.
Thanks. But I was asking about how to do the database search of double digested proteomes - do you set "enzyme" as trypsin? In Mascot you cannot select two enzymes - only one.
Posted: Tue Oct 14, 2014 6:18 am
Nothing is going to stick to an SCX material - or any other ion-exchange material - in the presence of molar concentrations of an electrolyte. It will outcompete the charged groups of your peptides and so will displace them from the stationary phase. Gd.HCl is an electrolyte.
I too have read that 5% trifluoroethanol denatures proteins nicely and leads to good digestion. This is an alternative to the addition of urea or Gd.HCl. Also, be advised that trifluoroethanol is extremely toxic.
Posted: Wed Oct 15, 2014 3:06 am
Just to add, it also really depends on the context. What kind of sample are you working with, what kind of protein amounts are you using, what kind of lysis buffer did you use, was there SDS in there ?
Posted: Thu Oct 16, 2014 1:43 am
m/z wrote:A link to a optimized protocol would be great.
Posted: Sun Oct 19, 2014 2:14 am
I agree with "tsbatth's" suggestion of using GuHCl instead of Urea for trypsin digestion. I would also recommend following publications in this regard. Kindly have a look and best regards.
Posted: Sun Oct 19, 2014 11:18 pm
@Kelstrup & Lucky: Thanks for the suggestions!
Posted: Fri Oct 31, 2014 4:22 am
We just published a paper describing very high identification rates using Kulak's ideas but in normal eppendorf tubes:
Also, there may be some protocols here that you will find useful?
PrimeXS protocol link
Posted: Wed Nov 05, 2014 12:07 pm
We just published a paper describing very high identification rates using Kulak's ideas but in normal eppendorf tubes:Link
Also, there may be some protocols here that you will find useful?PrimeXS protocol link