Hi,

I don't think that the ortho-phosphoric acid is the reason for the lack of peptides in your in gel digest, assuming that you performed some consecutive acetonitrile/trypsin digestion buffer washes of the excised gel plugs prior to adding the protease?

Do you observe some tryptic autodigestion fragments in your MALDI spectra? If not, then I think nothing is lost and you can just repeat the in gel digestion procedure. If you do see tryptic autodigestion fragments then the amount of protein in gel might be too low..

Hope this helps!

Thank u Eef...repeated the peocedure and performed some extra washing steps and modified the protocol a little.. got the peptides now.

ortho-phosphoric acid and methanol are fine for MALDI. I recommend you to try a control experiment with different concentration of a specific protein.