https://www.ncbi.nlm.nih.gov/pubmed/22002805

Urea buffer would be your best option;

https://www.thermofisher.com/nl/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/sample-preparation-mass-spectrometry.html

Dear Maynak,

I would solubilize the protein in 6-8 M Urea with Protease inhibitors, followed by reduction and alkylation.  After that step, you need to dilute down the urea to about 1M Urea since Trypsin is not very active in 6-8M Urea.  Add trypsin to have ~5ng/uL of digest solution.

There is a very good methods publication that you can follow - even if it is published with phosphoproteomics in mind, it is still a very good protocol to follow for solution digestions.

Good luck,

Hediye.

Dear Hediye,

Thank you so much. But, there are various papers that have showed that urea method is not suitable for mass-spec due to its incompatibility.Have you ever tried with sodium-deoxycholate?

After digestion, peptides can be cleaned up with OASIS HLB columns to get a clean peptide mix that is MS compatible, since the elution buffer of the OASIS columns is TFA & ACN.

Hi Maynak,

You have to do a peptide clean up step, post in-solution digestion.   You can use stage tips, C18 Zip Tips or Poros R50 hand packed tips.  If you clean up the peptides, urea method does not interfere with downstream LC-MS/MS steps.

Best,

Hediye.

@Hediye and Tim

Actually, I have read a paper where they mentioned about the differential effect of urea and SDC as lysis buffer reagent. I am a beginner in this field and thats why I am a little bit confused.I have attached it below.

Hi Maynak,

I have had a quick look at the attached publication by Leon et al.and specifically, highlighted areas that are points of your concern.

I have spent a fair amount of time and identified many unknown proteins during my career and a supporter of peptide clean up before any mass spectrometry step.  SDS-PAGE denatures and cleans up proteins prior to in-gel digestion.  Before the in-gel digestion, residual SDS can be removed from the gel matrix with Methanol washes.  After in-gel digestion, peptide pools are cleaned up and ready for LC-MS by making sure that you include a Sep-Pak, Stage Tip or C18 Zip tip, any kind of clean up step you are familiar with.  I do not recall having any interference with LC-MS/MS experiments once I follow these steps religiously.

For solution digests, urea is very effective in denaturing and solubilizing the proteins.  The only essential point I can make is that you should make up the ~6M Urea fresh, each time you need to use it.  Endoproteinase LysC works very well in high urea so you can take advantage of this by using LysC as first enzyme for 2-4 hours followed by dilution of the sample containing Urea down to ~1M to begin the trypsin digestion overnight. This method will give you the most efficient digestion.  However, you still need to clean up the generated peptides before any LC-MS/MS step. For total protein contents over ~10ug, you will need to use Sep-Pak cartridges.  Under 10ug, you can use Stage tips to clean up the peptides.

Good luck Maynak,

Best,

Hediye.

Thank you so much.