Chromatography Forum

I have what seems like it should be a relatively straight forward MS task- to ID a peptide (we believe exists) in insect heads. This ligand is > 80 aa and well retained on RP as well as cation exchange columns. pI near 8.

Main concerns are to minimize proteolytic degredation, and to use selective solubility to enrich sample. The peptide is soluble and extractable in 90/10 MeCN/HOAc, so I imagine this excludes most proteins much larger than 20 KDa. Also seems proteases, if soluble, ought to be inactive in such a solvent composition. However, lipid material is also extracted. This lipid material co-eluted (or escaped) SPE and fouled the prep C4 column.

I am considering liquid/liquid extraction of the MeCN/HOAc sup with hexane, or else, adding something miscible, such as DCM (acetone? MeOtBu?), hoping my target peptide will eventually precipitate.

Any rough idea as to the amount of DCM I may need to add (1-2, 10 volums?) before I should give up on precipitation, and add hexane and try liquid/liquid extraction?

I have what seems like it should be a relatively straight forward MS task- to ID a peptide (we believe exists) in insect heads. This ligand is > 80 aa and well retained on RP as well as cation exchange columns. pI near 8.

Main concerns are to minimize proteolytic degredation, and to use selective solubility to enrich sample. The peptide is soluble and extractable in 90/10 MeCN/HOAc, so I imagine this excludes most proteins much larger than 20 KDa. Also seems proteases, if soluble, ought to be inactive in such a solvent composition. However, lipid material is also extracted. This lipid material co-eluted (or escaped) SPE and fouled the prep C4 column.

I am considering liquid/liquid extraction of the MeCN/HOAc sup with hexane, or else, adding something miscible, such as DCM (acetone? MeOtBu?), hoping my target peptide will eventually precipitate.

Any rough idea as to the amount of DCM I may need to add (1-2, 10 volums?) before I should give up on precipitation, and add hexane and try liquid/liquid extraction?

1) Proteolitic degradation... you use proteolitic ferment (as pepsine) from destruction of biologic raw, protease in normal presentedin raw (as steptokinasa in hemolitic streptococcus)?
2) Lipids- its may not lipids, its may lipoproteids.This is worse than

I use protease inhibitors: AEBSF and peptstatin. I'm counting these, low pH, 90% MeCN, and ice-cold temp to minimize proteolysis.