****************************************************************************** Date: Tue, 09 Mar 1999 11:39:07 -0800 From: Deb McMillen Subject: MS method for distinguishing leu/ile Organization: * Hi, all, I have a group here that is routinely interested in determining if isoleucine or leucine is present in their recombinant proteins. I tell them that amino acid analysis is the way to go. They say they have heard of a method using mass spec that will allow this distinction--What I'd like to know is, is this true and how is it done? Thanks, Deb McMillen Institute of Molecular Biology University of Oregon Eugene OR 97403 ****************************************************************************** From: "M Sweeney - MSMS Consulting" Date: Wed, 10 Mar 1999 10:26:06 -0800 Subject: Re: MS method for distinguishing leu/ile Organization: EarthLink Network, Inc. This is a limiting problem for low energy CID mass spectrometers. They don't generate the side chain fragments required to differentiate the two species. Some groups have attempted to address this. I don't recall a trivial answer yet however... Try Differentiation of leucine and isoleucine residues in peptides by consecutive reaction mass spectrometry Nakamura et al., Anal Chem 1990 Feb 1;62(3):311-3 Novel fragmentation processes of peptides by CID in a tandem mass spectrometer: differentiation of leucine and isoleucine Johnson et al, Anal Chem 1987 1;59(21):2621-5 Identification of a new type of fragment ion in the collisional activation spectra of peptides allows leucine/isoleucine differentiation Stults et al, Biomed Environ Mass Spec 1987 Oct; 14(10:583-6 Differentiation between the isomeric amino acids leucine and isoleucine using low-energy CID tandem mass spectroscopy Hulst et al., J Mass Spec 1996 Oct;31(10):1188-90 A simple method for differentiating Leu and Ile in peptides. The negative ion mass spectra of [M-H]- ions of phenylthiohydantoin Leu and Ile. Ramsey et al, Rapid Commun Mass Spectrom 1995; 9(13):1241-3 An approach to differentiation of leucine and isoleucine residues in EI mass spectra of peptides (1) Waern et al, Biochem Biophys Res Commun 1978 Mar 30;81(2):448-54 Mass Spectrometric differentiation of leucine and isoleucine in proteins derived from bacteria or cell culture Herihy et al, Biochem Biophys Res Commun 1981 Sep 16; 102(1):335-40 I don't know of any groups that rely on MS (Quads, Traps, or TOF instruments) to determine the difference between these two as a primary method. It is generally considered a blind spot for MS that would benefit from a method and so there are a variety of papers discussing observations on this system. Higher energy CID process can generate diagnostic fragments. A larger peptide MS/MS spectra would have a lot of peaks at High CID and one or two of the little ones would be the sign you look for. So it is not THAT routine. At least that has been my perception. Although for some it may be. mattsweeney@earthlink.net Mass Spec Consulting Training/Operations/Consulting/Method Development LC/MS Pharmacokinetics, Peptides, Proteins, Metabolism, Maintenance Classes, Specialist in Finnigan Equipment and Software ****************************************************************************** From: gal@taloa.unice.fr (J.-F. Gal) Date: Mon, 15 Mar 1999 14:17:10 +0000 Subject: MS method for distinguishing leu/ile Organization: * Hi Deb, You can use derivatization and GC (or GC/MS) with acetyl chloride (on N) and propanol (on acid side) for example, they are rather well resolved on low polarity columns. Cheers, Jean-François ------------------------------------- Prof. J.-F. GAL Groupe FT-ICR*GRECFO-Chim. Phys. Org. Fac. des Sciences*Parc Valrose Univ. de Nice-Sophia Antipolis 06108 NICE Cedex 2*FRANCE Tel : (33) (0)4 92 07 61 10 Fax : (33) (0)4 92 07 61 11 E-mail : gal@unice.fr -------------------------------------