Message-Id: <v02140b04af0eb004cac1@[134.197.50.216]>
Date: Fri, 24 Jan 1997 10:32:19 -0800
From: schooley@unr.edu (David A. Schooley)
Subject: Re: ProtSeq:Feasibility of MS Detection for Edman Sequencer
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Greg-
Check out paper below. It seems you are referring to doing no HPLC
separation? Sounds like tough order! As long as you still do HPLC
separation Leu/Ile descrimination should be no problem. Check out this
reference:
Aebersold R., Bures E. J., Namchuk M., Goghari M. H., Shushan B. and Covey
T. C. (1992) Design, Synthesis, and Characterization of a Protein
Sequencing Reagent Yielding Amino Acid Derivatives with Enhanced
Detectability by Mass Spectrometry. Protein Sci. 1, 494-503,
We report the design, chemical synthesis, and structural and functional
characterization of a novel reagent for protein sequence analysis by the
Edman degradation, yielding amino acid derivatives rapidly detectable at
high sensitivity by ion-evaporation mass spectrometry. We demonstrate that
the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is
chemically stable and shows coupling and cyclization/cleavage yields
comparable to phenylisothiocyanate, the standard reagent in chemical
sequence analysis, under conditions typically encountered in manual or
automated sequence analysis. Amino acid derivatives generated with this
reagent were detectable by ion-evaporation mass spectrometry at the
subfemtomole sensitivity level at a pace of one sample per minute.
Furthermore, derivatives were identified by their mass, thus permitting the
rapid and highly sensitive determination of the molecular nature of
modified amino acids. Derivatives of amino acids with acidic, basic, polar,
or hydrophobic side chains were reproducibly detectable at comparable
sensitivities. The polar nature of the reagent required covalent
immobilization of polypeptides prior to automated sequence analysis. This
reagent, used in automated sequence analysis, has the potential for
overcoming the limitations in sensitivity, speed, and the ability to
characterize modified amino acid residues inherent in the chemical
sequencing methods that are currently used.
David
David A. Schooley
Dept. of Biochemistry/330
Univ. of Nevada
Reno, NV 89557
schooley@med.unr.edu
tel: (702) 784-4136; fax (702) 784-1419