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Subject: Re: enzymatic digestions of proteins hplc separations
Date: Wed, 16 May 2001 11:54:12 +1000
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Dario,
you didn't mention that you have reduced or reduced and alkylated your BSA
before digestion. I seem to recall that there are 17 disulphides in BSA so I
suspect that your digest might contain some rather large, disulphide linked
peptides that might not elute from your LC column?
I would make a stock solution of BSA reduced and alkylated (I would use
acrylamide although others will argue for alternative alkylating reagents)
and use this in your control digests, you will be surprised by the
difference in digestion yield.
I used to keep chromatograms of native and reduced/alkylated HSA digests up
on the lab wall (these are strikingly different) to remind students of the
importance of thinking about cysteines before digestion.
Good luck....Ken
----- Original Message -----
From: "Dario Miranda" <dmiranda@burnham-inst.org>
To: "Recipients of ABRF List" <abrf@aecom.yu.edu>
Sent: Wednesday, May 16, 2001 2:59 AM
Subject: enzymatic digestions of proteins hplc separations
> Fellow listers,
>
>
> Chromatographers and proteomics laboratories:
>
> I work in the Molecular Analysis Laboratory of the Burnham Institute in La
> Jolla Ca.
> We are primarily an analytical protein characterization lab and a core
> service facility for the Institute. In preparation of mating my hplc to
> the triple-quad mass spec in the lab, I have been attempting to reproduce
> reverse-phase hplc separations of enzymatic digests of proteins. I seem
to
> be having sensitivity problems. I have begun using digests produced from
> immobilized-trypsin cartridges as I believe that the varying amounts of
> protein digested from an overnight enzymatic digest are not an adequate
> loading amount onto my column (usually about 5pmols). With the
immobilized
> trypsin cartridge I can digest discrete amounts of protein. Here are my
> conditions:
>
> HP1100 series hplc system using classic reversed-phase solvents (A= water
> with 0.05%TFA, B=80%Acetonitrile with 0.045% TFA) with LC packings 1:100
> flowsplitter, LC Packings U-Z view capillary flowcell (35nL volume, 8mm
> pathlength), HP1100 series variable wavelength detector (calibrated and
> used extensively at 205nm but a few runs at 195nm don't show any
> improvement in response), a new 180 micron inner-diameter 150mm long
> capillary column packed with LC Packings' own "pepmap' C18 solid phase (5
> micron pore size). The lamp has just under 1200 hours on it, but I don't
> believe that its useful life is over yet. I'm checking the intensity of
> it now.
>
> Initially, I was using tryptic in-gel digests of BSA, but it seemed there
> weren't enough peptides to give much more response than just a few
> unresolved peaks over the course of a 1-hour separation method (2-32%B in
> 30 min, 32-67% B in 45 min, 67-82% B in 60min. flowrate=4uL at the
column).
> The peptides are resuspended in 10uL of 2%TFA & 5% Formic acid after being
> completely dried. Maldi analysis of the in-gel digests has shown that
the
> digest HAS worked and a decent peptide mass-map can be produced from the
> spectra of the BSA digest. The chromatographic peaks I am getting are few
> and far between and seem to be barely in the detectable range.
> Assuming the the amounts of material produced by the in-gel digests has
> great variability, I switched to digesting BSA using an immobilized
trypsin
> cartridge. These can be produced fairly rapidly and also give comparable
> MALDI spectra. But my luck hasn't been much better.
> I have loaded upwards of 30 pmols of digested BSA onto this column and
have
> not been able to reproduce the resolution on the scale that I have seen
and
> read about in the literature.
>
> Does anyone have any ideas or references they can point me to? My only
> thought is that I might try a column heater and a temperature of ~35 C or
> so. Suggestions would be appreciated and I will tally my responses so
that
> I can pass on information that has worked for me.
>
> Thanks,
>
> Dario Miranda
> Molecular Analysis
> The Burnham Institute
> 10901 N. Torrey Pines rd.
> La Jolla Ca. 92037
> dmiranda@burnham.org
> 1-858-646-3100 ext.3991
>
>
>
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