Re: cys derivatization

Richard S. Johnson (rjohnson@immunex.com)
Thu, 23 Jul 98 11:17:17 -0800

Kathy:
My experience w/ the lcq is that if I can pick the correct m/z for
the precursor w/ s/n of one, I can get a decent msms spectrum. The problem has
been finding the ion. How have you been doing it? At the very low levels can
you actually see a s/n greater than one? I have to do 10 "zoom scans" over a
100 Da range, and look for doubly charged ions. I repeat this over
several 100 Da (note the use of Da, since I'm addressing a biochemist)
ranges - 500-600, 600-700, 700-800. All of this is done with nanospray;
are you doing nanospray or lcms? As for keratin, Gary H had mentioned
phosphine reductants, since you can use a lower concentration (and thereby
reduce the amount of contaminants). I think you already are using a
laminar flow hood, and that you avoid sticking your fingers in the vinyl
pyridine.

Rich

______________________________ Reply Separator
_________________________________
Subject: cys derivatization
Author: <Katheryn.Resing@Colorado.EDU (Katheryn Resing) > at Internet
Date: 7/23/98 10:01 AM

We have just gotten an LCQ and have found it has a sensitivity sufficient
to analyze in-gel digests of proteins that are VERY WEAKLY silver stained
(i.e, we can use fluorescent stains or radiolabeled samples). This means
that the keratin contamination caused by reduction with DTT or
mercaptoethanol prior to alkylation is now a serious problem. What
alternative protocols and tricks are being tried out there in ABRF land to
step down in sensitivity for in-gel digests (untried suggestions are also
welcome)?

Katheryn Resing