we have the following problem. We are trying to find phosphorylation sites
in a small protein. We know that the sites are clustered in a highly basic
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, so
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 low
background spectra. So far we worked with ammonium bicarbonate, but the
results weren't very convincing. What buffers would the experts recommend
for best spraying and cleanest background? Any input is highly
appreciated.
Best regards
Paul Jenoe
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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
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