Tryptophan analysis from PAGE hydrosylate

Tony Larson trl1 at york.ac.uk
Wed Jun 6 03:49:12 EST 2001


I'm looking for advice on the best way to hydrolyze a protein band cut out
from a PAGE gel to its component amino acids, and then to derivatize and 
analyze these amino acids by HPLC or GCMS.  One of my main concerns is that
that tryptophan and hydroxyproline are major constituents of the protein in question,
so I don't want to lose these in the hydrolysis or derivatization procedures.
My initial thought is that I would need to use methanesulfonic acid hydrolysis
(rather than HCl) to preserve the tryptophan, but as MSA is not volatile, I am
unsure how I should treat the hydrosylate for derivatization.  To summarize, I'd
like to know:

1) Are there any special procedures needed for hydrolysis of excised PAGE gel slices?
(especially with MSA as opposed to HCl)

2) Are there background contamination issues with gel hydrosylates?

3) Is there an amino acid derivatizing procedure compatible with residual MSA?
(I've used OPA, PTC, and DABS-Cl methods for HPLC and TMS derivatives
for GCMS of dried amino acids).

4) Is there a procedure for partitioning amino acids away from MSA hydrolyzed
protein?

thanks
Tony




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