We have been trying to develop an Amino Quant method for AAA of peptides
solubilized in DMSO and have had mixed success. We are aware of the
previous discussions on the impact of residual DMSO during acid hydrolysis
(ABRF e-discussions held Nov 1998;
http://www.abrf.org/archives/hmail/9832/0071.html). We have two major
problems.
We are experiencing this effect carrying over into vials of standards that
do not contain DMSO (e.g. NIST BSA and NIST amino acids) that are in the
same gas space during hydrolysis as the DMSO-containing samples. We are
drying less than 10 ul of DMSO -peptide solution for almost an hour under
speedvac, so we felt the drying was sufficient. Question #1: Is there
possibility that enough residual DMSO is present to enter the gas phase and
nuke residues in other, adjacent vials?
In addition to the previously-noted effects on TYR, MET, HIS, and somewhat
SER, THR (PRO is already poor due to AminoQuant chemistry), we have seen an
impact on GLY in some runs. Even in free NIST amino acids, GLY peak area is
increased in some injections, although resolution between the adjacent
residues of HIS and THR is quite good (so no shoulders, etc... to affect
integration). Question #2: Is there some effect of DMSO on GLY, too? Or
is there something (oxidation product? artifact of DMSO + FMOC/OPA
derivatization chemistry?) that could be co-eluting with GLY to increase
peak area in some injections? There is no GLY in the sample buffer, so no
variable residual GLY should be present, and even if it were, this effect is
seen in the NIST AAs too.
Any ideas? Note: We do not currently have the option to run these samples
on any AAA system (ie ninhydrin, Accutag, PTC) other than Amino Quant, and
we use a Savant Hydrolyzer system that employs one common gas space for all
samples, although we are switching to PicoTag workstation and Mininert vials
soon.
Thanks in advance
Nadine Ritter
BioReliance
nritter@bioreliance.com
This archive was generated by hypermail 2b29 : Fri Jun 30 2000 - 07:51:55 EDT