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J Biomol Tech. 2007 February; 18(1): 33.
PMCID: PMC2291886

P96-T Characterization the Sequence and Post-Translational Modification of Large Peptides by Tandem ETD MS/MS


Protein/peptide analysis is commonly based on use of a tandem mass spectrometer combining electrospray ionization and collision-induced dissociation (CID). The major disadvantage of collisional ion activation is the internal heating of the parent ion, which predominantly yields cleavages of the weakest bonds, resulting in less informative fragment spectra; this often limits peptide sequence determination.

To better elucidate the peptide structure, more selective fragmentation techniques are of huge interest. Electron transfer dissociation (ETD) in a non-linear Paul trap has been introduced as a new fragmentation technique, which avoids internal parent ion heating. Induced by the electron transfer, the intermediate peptide radical cations fragment randomly at each amino acid position of the peptide backbone, which is particularly suitable for the sequence characterization of larger peptides and for post-translational modification (PTM) identification.

We investigated the electron transfer dissociation of large peptides using the Bruker HCTultra PTM Discovery System. Multiply charged positive peptide target ions are generated via conventional electrospray or via off-line nanospray. Electron transfer dissociation of larger peptides is done in a non-linear three-dimensional Paul trap, where consecutive trapping of peptide cations as well as reagent anion accumulation is enabled under full automatic software control.

Common resonance precursor activation via CID MS/ MS almost always results in poor sequence information if the peptide molecular weight exceeds 3000 Da. ETD MS/MS of the same precursor ion provides much better sequence information. Based on the enhanced resolution of the utilized instrument, since even quadruply charged fragment ions are identified, the entire amino acid sequence is read out of the ETD MS/MS spectrum.

Even when larger PTM peptides were investigated under ETD conditions, labile bounded PTMs remain attached to the peptide backbone, and are localized without ambiguity. ETD MS/MS data of larger phosphorylated as well as glycosylated peptides will be presented.

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