Chris -
You can isolate a blocked N-terminal peptide from a tryptic digest using a
strong cation-exchange column. At pH 2.7-3.0, the typical tryptic peptide
has a charge of +2, due to the N-terminus at one end and the Lys- or Arg-
residue at the other end. Peptides with His- residues have higher + charge,
and disulfide-linked peptides have a charge of +4 (making them easy to
isolate selectively). Now, the C-terminal peptide has a charge of +1 (unless
the C-terminal residue is Lys- or Arg-). That means it will be the first or
among the first peptides to elute in a salt gradient. Gorman and Shiell (J.
Chromatogr. 646 (1993) 193) used our PolySULFOETHYL Aspartamide columns in
this manner to isolate the C-terminal peptides from a number of tryptic
digests. Similarly, a blocked N-terminal tryptic peptide also has a charge
of +1 and is the first or among the first peptides to elute from an SCX
column; see Kawasaki et al., J. Biochem. 102 (1987) 393.
So - get a suitable SCX column and run a salt gradient at pH 2.7. Your
N-blocked peptide will be among the first 3 peptides to elute.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: (410) 992-5400 FAX: (410) 730-8340
*******************************************
<< Subj: Protein N-terminus
Date: 08/25/2000 8:06:14 PM Eastern Daylight Time
From: turck@itsa.ucsf.edu (Christoph W. Turck)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
We want to determine the N-terminal amino acid sequence of a protein.
Edman degradation did not yield any results, so we suspect a blocked
N-terminus. Are there any methods determining the N-terminus using mass
spectrometry? How likely is it that one finds the N-terminal tryptic
peptide of the protein after an in-gel digest? Or are there any tricks that
one can use to increase one's chances of finding the N-terminal peptide?
Thanks,
Chris (turck@itsa.ucsf.edu)
This archive was generated by hypermail 2b29 : Fri Sep 01 2000 - 16:10:38 EDT