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J Biomol Tech. 2010 September; 21(3 Suppl): S56.
PMCID: PMC2918149

Complete Protein Characterization using ETD and PTR with the Combination of Top-Down and Bottom-Up Strategies in an Ion Trap

A. Ingendoh,2 R. Hartmer,2 A. Brekenfeld,2 A. Schneider,2 and A. Kiehne2
1Bruker Daltonics Inc., Fremont, CA, United States;
2Bruker Daltonik GmbH, Bremen, Germany

Abstract

RP-111

Collision induced dissociation (CID) has drawbacks in terms of a limited applicable molecular weight and the dependence of the fragmentation efficiency on the individual bond strength and amino acid sequence. Electron transfer dissociation (ETD) and its related proton transfer reaction (PTR) are alternative ways of fragmenting peptides and proteins.ETD became the preferred method for the analysis of common post-translational modifications (PTM) in proteins since it preserves the bonds of modifications. ETD in combination with PTR is a most useful experiment for the fragmentation of larger peptides or even small proteins in the top-down approach. Intact proteins can be identified and/or sequenced without any prior enzymatic digestion. The measurements were done on a Bruker amazon ion trap. Fluoranthene is used both as ETD and PTR reactant. Switching nCI parameters decides if the radical anion for ETD or a basic anion for PTR is extracted from the nCI source. The PTR-reagent as well as the ETD-reagent are generated at an appropriate ionization chamber voltage. For mixed and multiple modifications on the same tryptic peptide CID does not give useful information, especially, if all modifications result in neutral losses during CID. This is the case for the combination of carbamidomethylation of Methionin (a sample preparation artefact) with phosphorylation. With ETD, all modifications stay intact on the peptide backbone and unambiguous assignment is possible. Presented here will be data on various modifications, including the differentiation of the isomeric amino acids aspartic acid and isoaspartic acid. Furthermore, top-down terminal sequencing of intact proteins and localization of cystein disulfide bridges will be shown.


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