Dear all,

Commonly, people use ACN to precipitate proteins out of plasma sample and then inject the supernatant to LC-MS/MS. This procedure is called "PPE-protein precipitation extraction".

The question is, when ACN is added to plasma, what exactly are those precipitates? Proteins? If so, how come proteomics people can inject protein digested fragments and use reversed phase LC to separate them (Water/ACN)? Why the ACN in mobile phase does not precipitate the digested proteins? Plus, I have seen people use RPLC(H2O/ACN) to separate intact proteins too....

Any comments?

Protein precipitation does not remove all the protein, it just removes enough to avoid a clogging of the column. If you look at a protein precipitated sample in the UV, and run a reversed-phase gradient, you see plenty of peaks, and I don't think that they are all small molecules.
While protein precipitation is simple, it does not clean a sample as well as a well designed SPE procedure.

In Plasma/Serum you have about 7% by weight protein, when you do RP of proteins you may use µg or ng of proteins. Still, if you have the wrong proteins (or too high an ACN conc.) you might get precipitation (or more likely, a strong adherence to the stat phase) due to the ACN, etc.

Now, to precipitate about 98% of plasmw/serum proteins should be quite good, that means you still have ~2g/100mL, that could be quite a problem.
We have had problems with SPE as well, apparently including problems due to proteins, and needed two pre-columns (one a 250x4.6mm the other a 20x2mm column) to get reasonable results (we prefer to call this a three step HPLC, rather than three dimensional). Just depends on what you are doing.

A simple answer to your question is that in general high concentrations of acetonitrile are used to precipitate proteins when for example analysing small molecules in blood. The concentrations of acetonitrile used for the analysis of proteins themselves in RP-HPLC are generally much lower. As you state in your question itself, much of the RP-proteomics work concentrates on peptides and small proteins, which are also less likely to preciptate than the larger proteins. I believe even for smaller proteins there may be loss of enzyme activity due to conformational changes which take place during the separation. For larger proteins, and I guess very hydrophobic proteins which would stick fast to a RP surface and thus require more acetonitrile to get them off (which could cause them to precipitate) then techniques which do not require organic solvents (e.g. ion-exchange and gel filtration can be used).

David