I second Kathryn's suggestion of looking for a higher carbon load C18 res=
in
for your chromatography, we have had some luck with the Inertsil (TM)
media, with "Ultra" endcapping and an 18.5% phase loading, we have been
able to trap polar dipeptides on columns packed by SGE.
Personally though, I would be throwing a few other proteases at the
protein. I know you say you want to use lys-c but, as with most
phosphorylation sites, the production of small polar peptides is a common
occurence when using basic residue directed proteases. Why not try Asp-N =
or
even thermolysin or chymotrypsin?
If you do want to infuse the digest directly, I would have thought ammoni=
um
acetate would be your best bet but the chances of your small polar peptid=
es
being "invisible" in the total digest would be high unless you were
planning on doing negative precursor-ion scanning in which case you need
your digest in alkaline conditions anyway?
Regards...Ken
>Dear ABRFers,
>
>we have the following problem. We are trying to find phosphorylation sit=
es
>in a small protein. We know that the sites are clustered in a highly bas=
ic
>stretch of the protein, but we do not know the precise location of the
>sites yet. When the protein is cleaved with trypsin or endoproteinase
>LysC, extremely polar peptides are released. We tried to feed them into
>the MS via capillary reverse-phase columns, but the peptides are lost in
>the flow through. We now want to cleave the protein and infuse the
>cleavage products directly, so that we do not lose the polar
>phosphopeptides. We isolate the protein from a reverse-phase column, it =
is
>then in 0.1% TFA, containing approx. 25% acetonitrile. What are the
>experiences in the ABRF community with respect to buffer compositions, s=
o
>that the digested protein is immediately ready for infusion? We want to
>use endoproteinase LysC for digestion. Obviously, one needs a buffer to
>adjust the pH to around 8, and the buffer should be volatile and give lo=
w
>background spectra. So far we worked with ammonium bicarbonate, but the
>results weren't very convincing. What buffers would the experts recommen=
d
>for best spraying and cleanest background? Any input is highly
>appreciated.
>
>Best regards
>
>Paul Jenoe
>
>=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D
>
>Paul Jenoe, PhD
>Department of Biochemistry
>Biozentrum of the University of Basel
>Klingelbergstrasse 70
>CH-4056 Basel
>Switzerland
>
>Tel. ++41 61 267 21 68
>Fax. ++41 61 267 21 48
>e-mail jenoe@ubaclu.unibas.ch
>
>=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=
=3D=3D=0D=0D=9D
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Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
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