Norman--
I'm assuming you're using acetonitrile as your organic solvent. Sometimes
switching to a MeOH/H2O (w/ H+ of course) solvent system can provide the
change in separation that you want. (MeOH is a "weaker" solvent.) Also,
you can try formic acid instead of acetic acid as your modifier; use less of
it, though--about 1%.
Good luck!
Kara Pearson
NHLBI/NIH
Bethesda, MD 20892
301-435-4490
>From: Norman Watts <WATTS@calvin.niams.nih.gov>
>To: Recipients of ABRF List <abrf@aecom.yu.edu>
>Subject: MS, better reversed phase solvent
>Date: Fri, 29 Sep 2000 11:47:35 -0500 (EST)
>
>I am attempting to follow phosphorylation of a protein by
>ES-MS on a HP1100 fitted with a Zorbax 300 SB-C3.
>
>I have two problems, resolution on the column and sample
>solubility.
>
>1) The resolution problem is that the phosphorylated peak
>is not as well separated from the non-phosphorylated
>material as I would like. Can anyone suggest something
>other than making the gradient even more shallow.
>
>2) I need to scale up a bit because I am working with a
>very small amount of protein. However, I know the protein
>both polymerizes and aggregates above a few 10s of ug/ml.
>I suspect that the 10% HAc is just not sufficient and so
>most of the sample comes off in the flow-thru. I am
>thinking of adding 4 M urea. Can anyone suggest a better
>solvent?
>
>
>thanks,
>
>norman
>
>------------------------------------------------------------
>
>Norman Watts, PhD.
>Laboratory of Structural Biology
>NIAMS, National Institutes of Health
>Bethesda, MD 20892-2717
>U.S.A.
>E-mail: watts@calvin.niams.nih.gov
>Phone: 301-402-3418
>Fax: 301-480-7629
>
>------------------------------------------------------------
>
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