I have precipitated proteins using Methanol:chloroform method, and thus obtained pellet has been solubilized using 8M urea for quantification purpose.
Now i want to analyze/ identify proteins using LC-MS (after Tryptic digestion).
Thus i want to know whether any one has come across such situation, then kindly give input.
Appreciation in advance...
In general no, but it depends on much you're injecting into the LCMS. But you must be sure you use a volatile pH buffer like formic acid (not phosphate!).
Hi, Shridhar Chougule,
Any salt influence in the case of big concentration, it is especially for the protein with the big molecular weight and less for peptides. In the solution with the big concentration of salt pik of big proteins became smaler and a little wider. You could find some article on this theme if You want.
Hi Shridhar,
It is a good practice to desalt the peptides before LC-MS/MS analysis. For peptide amounts ~less than 5ug, you can use double layer of Stage tips to carry out the desalting. If you have peptide amounts that are 10ug and above, you will need to use SepPak columns to desalt your peptides. The desalted peptides are in a volatile solvent composing of acetonitrile and formic acid which can be removed by speed vac step. After speed vac, you can reconstitute/dissolve the purified peptides; there will be no urea left to interfere with the LC-MS/MS step.
Good luck,
Hediye.
Urea in aqueous solution is in equilibrium with ammonium cyanate. The cyanate can carbamylate primary amino-groups in Lys and at the N-terminus. This would be detectable as an additional peak in MS. To prevent this, simply use only freshly prepared urea solutions.
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