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J Biomol Tech. 2007 February; 18(1): 34–35.
PMCID: PMC2291844

P101-M New Setup for Top-Down Characterization of Proteins via Consecutive Ion/Ion Reactions

Abstract

The conventional MS “bottom up” strategy for protein identification is the tandem MS/MS analysis of tryptic digests of complex proteins mixtures. Via enzymatic digestion, even larger proteins are cut into smaller peptides. In order to reduce the resulting complexity of the tryptic digest, additional chromatographic separation is used in front of the tandem MS/MS analysis. The bottom-up strategy, however, has shown its limitation when the characterization of multiply post-translational modified protein is of interest. The unique identification of several individual peptides from a tryptic digest does not provide the needed information if the interaction of the different modifications of the intact protein is of primary interest.

Electron transfer dissociation (ETD) is particularly dedicated for the sequence analysis of larger peptides as well as for the identification of PTMs, as weakly bonded PTMs survive the prompt odd-electron-driven fragmentation of the electron transfer. However, if a multiply charged protein (z > 10) is analyzed via tandem ETD MS/MS, the resulting fragmentation spectrum shows a highly complex mixture of multiply charged fragment ions (zfragment ion = 1–9).

We have modified the nCI source as well as the acquisition software of the HCTultra PTM Discovery System to allow the selective and consecutive ion accumulation of different types of reagent anion. For the initial ETD step, odd-electron reagent anions are selectively accumulated, and for the subsequent proton-transfer reaction, even-electron reagent anions revealing a strong proton affinity are accumulated. The second PTR step is necessary to significantly reduce the complexity of the above-mentioned ETD MS/MS-data, particularly when highly multiply charged proteins are investigated. The final charge stripping step produce a mixture of 1+, 2+, 3+, and 4+ fragment ions. With the present setup we were able to characterize the amino acid sequence of the intact proteins (e.g., ubiquitine, cytochrome C).


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