Pre fractionization- CHCl3/MeOH, phenol

[DJ]

I am trying to isolate a few basic peptides between 3-8,000 Da from brain tissue. I have previously extracted lyopholized tissue with 90/10 MeOH/acetic acid, centrifuge, evaporate supernatant, then put through C4 SPE. However, I'm looking into additional ways to clean up the sample before LC.

Chloroform/MeOH is attractive since it might minimize acidic protease digestion (that may occur with acidic methanol), however, use of CM seems a little unorthodox (compared to widely used MeOH/HOAc)

Does anyone have experience using various extraction protocols with organics, such as chloroform/MeOH, or phenol, in attempts to remove as much non-specific protein/cell constituents as possible before multi-demensional HPLC?

Would use of such organics be useful for selectively separating higher MW proteins from peptides?

[HW Mueller]

Among other things there is a huge work on handling nerve tissue. If memory does not fail me it was called "Handbook of Neurochemistry". Generally, stopping enzymatic reactions with extraction solvents is an action which comes way too late.
There are also extensive monographs on protein purification and handling which all are good starting points for any analytical work of this highly complicated type.

[kerri]

You can also perform ACN precipitation. C/M, M/acid, and ACN, ACN/acid will all precipitate the larger proteins out. I see you are using 10% acetic acid in your ppt. That's a lot. It will contribute to the time spent in the evaporator and it might not be necessary. You could try to use less of a stronger acid, like formic acid or even TFA (the latter is useful if your compounds of interest are compatible).

Any of above methods will work depending on if the smaller proteins you are interested in will dissociate from the larger proteins and avoid being precipitated out as well. The addition of acid can helps by partially/fully denaturing the large proteins, thereby releasing the ligands or small-bound drugs. Organic will ppt the majority of the proteins. You can play around with the ratios of organic to matrix (v/v) to get the results that work best.

A good paper: Polson, C, et al. “Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry.â€