CABELL.ANDREWS@BIONET-20.ARPA (05/13/88)
From: Paul Andrews <CABELL.ANDREWS@BIONET-20.ARPA>
RE:Blocked N-terminal peptides
I am interested in retrieving a blocked N-terminal peptide from a
mixture of peptide fragments from a trypic digest of a membrane protein. I
already have the deduced amino acid sequence of it and want to confirm that I
have the correct starting point. I can envision using two HPLC columns in
sequence. The first would be a cation exchange column which would be coupled
directly to a reverse phase column (C-18 or even C-8). and by acidifying
the tryptic digest and eluting initially with water only (0.05% TFA) the
vast majority of the peptides would be retained on the first column because they
would be doubly charged (Trypsin cleaves at Arg and Lys!!). The blocked
N-terminal peptides as well as the COOH terminal peptides (plus a few peptides
more probably) would go through the cation exchange column in the void volume
and be retained on the reverse phase column. These unretained peptides could
then be eluted with a typical water/ACN (0.05% TFA) gradient.
Has anyone out there attempted anything like this? I don't really
relish the idea of fishing around in a tryptic digest looking for a peptide
that I can't get any gas phase sequence data before I start doing MS/MS on it.
I suppose I could always try to hang some kind of chromophore or radioactive
label on an amino acid that would be in the blocked N-terminal peptide but
that would start to create problems in the MS/MS analysis.
Paul Andrews
(CABELL.ANDREWS)
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