The following procedure was what I used exclusively to prepare
polypeptides isolated in 0.1%TFA/40% acetonitrile for both LysC and
tryptic mapping. SpeedVac the reversed-phase pool to 1/10 the original
volume. (CAUTION: The trap must be empty prior to concentration in
order to get the acid concentration sufficiently low.) Then, to a
defined volume of concentrate, add 1/5-volume of 1M Tris, pH 8.5. Check
the pH of this mixture; ideally, it should be around 8.3. Both LysC and
trypsin should work well under these conditions. Although you don't
want to electrospray the Tris, the completed digest could be
buffer-exchanged into a volatile solvent, using ion-exchange (e.g.,
Microcon-SCX) or HILIC. I haven't tried using a concentrated volatile
buffer in place of Tris; if you could find one which still gets the pH
of the concentrated protein high enough for digestion, you could
eliminate the buffer exchange step and go right into the mass spec.
Mike Klein
Amgen, Inc.
> ----------
> From: jenoe@ubaclu.unibas.ch[SMTP:jenoe@ubaclu.unibas.ch]
> Sent: Tuesday, June 30, 1998 2:39 AM
> To: Recipients of ABRF List
> Cc: paul jenoe
> Subject: Digest/MS/Infusion
>
> Dear ABRFers,
>
> 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
>
> ======================================================================
> ======
>
> 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
>
> ======================================================================
> ======
>
>