This happens all the time with in-solution digests, I believe it is the trypsin itself coming out of solution, or larger peptide fragments. Precipitate formation is increased by incubating on ice, just do this then centrifuge away the precipitate. It should not affect your peptide yield. We, and everyone in our department, has precipitates form upon acidification of in-solution digests. You should stage-tip (C18) purifify your peptides before running them on the MS anyway.

One thing I would recommend is to use 0.1% Formic acid not 1% for nLC-MS/MS. In my experience that high of a concentration of FA will bias your peptide ionization to mostly the +3 and +4 charge states when in any experiment the most abundant species will be +2. 

Than being said, can you please send details of your digestion protocol so I can better answer your question. 

Thanks

I will try to reduce the FA and I will also use a fresh stock of trypsin.

My protocol involves protein solvation (8M Urea/20mM methylamine), reduction with DTT (10 mM final), alkylation with IAA (22 mM final), sample dilution to reduce the Urea to 1 M (50mM Tris/1mM CaCl2) and addition of 2 ug Trypsin and incubation over night.

To measure I dilute my sample with ACN and FA .

thanks four your ideas

I see several things here. 1) use only Urea not methylamine, 2) IAA at 22 mM may be too high and will lead to overalkylation of peptides creating artificial modifications, that will not be detected in your database search and also skew your quantitation. What it the total amount of protein you are digesting? I can get a better idea is the IAA is too much if I know the protein amount. 3) Tris and CaCl2 precipiates in low pH conditions, like 1% FA. So I recommend that you ziptip your samples first, then use the nLC-MS buffer at 0.1%. You can also switch to ammonium bicarbonate buffer (I can give more details on this if you want). 

Also what kind of sample are you trying to digest?

I am digesting E coli lysate and  I use about 100 ug protein. If you think its the Tris buffer I will give ammonium bicarbonate buffer a try. Of course it would be nice if you can hand me more details.

I just checked, my Tris buffer is stable upon the addition of FA so must be something else.

Hmmm E.coli should not have this problem. 

1) 22 mM IAA is ok for 100 ug of digest. 

2) I would still remove the methyl amine.

3) I use 25 mM ammonium bicarbonate as my digestion buffer. Tris precipitates in low pH and cold temp. (e.g. +4C in the autosampler).

4) Since it may be your trypsin digest is not complete, you can first digest with Lys-C (1:100 w/w) overnight at 37C since it can tolerate 8 M urea.  After that dilute to 1 M urea and use trypsin (1:50 w/w) overnight at 37C. This should help get rid of big peptide fragments.

Good luck, 

Norelle

This happens all the time with in-solution digests, I believe it is the trypsin itself coming out of solution, or larger peptide fragments. Precipitate formation is increased by incubating on ice, just do this then centrifuge away the precipitate. It should not affect your peptide yield. We, and everyone in our department, has precipitates form upon acidification of in-solution digests. You should stage-tip (C18) purifify your peptides before running them on the MS anyway.

What is role of methylamine with urea? How use of methylamine is beneficial?

It also happened in my case. There is nothing to worry for that. But 0.1% - 1% FA is more reasonable than 2% FA. I agree with that.