uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Determination of the location of positive charges in gas-phase polypeptide polycations by tandem mass spectrometry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, MMS, Medical Mass Spectrometry.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
2006 (English)In: International Journal of Mass Spectrometry, ISSN 1387-3806, E-ISSN 1873-2798, Vol. 252, no 3, 204-212 p.Article in journal (Refereed) Published
Abstract [en]

Location of protonated sites in electrospray-ionized gas-phase peptides and proteins was performed with tandem mass spectrometry using ion activation by both electron capture dissociation (ECD) and collisional activation dissociation (CAD). Charge-carrying sites were assigned based on the increment in the charge state of fragment ions compared to that of the previous fragment in the same series. The property of ECD to neutralize preferentially the least basic site was confirmed by the analysis of three thousand ECD mass spectra of doubly charged tryptic peptides. Multiply charged cations of bradykinin, neurotensin and melittin were studied in detail. For n+ precursors, ECD revealed the positions of (n − 1) most basic sites, while CAD could in principle locate all n charges. However, ECD introduced minimal proton mobilization and produced more conclusive data than CAD, for which N- and C-terminal data often disagreed. Consistent with the dominance of one charge conformer and its preservation in ECD, the average charge states of complementary fragments of n+ ions almost always added up to (n − 1)+, while the similar figure in CAD often deviated from n+, indicating extensive charge isomerization under collisional excitation. For bradykinin and neurotensin, the charge assignments were largely in agreement with the intrinsic gas-phase basicity of the respective amino acid residues. For melittin ions in higher charge states, ECD revealed the charging at both intrinsically basic as well as at less basic residues, which was attributed to charge sharing with other groups due to the presence of secondary and higher order structures in this larger polypeptide.

Place, publisher, year, edition, pages
2006. Vol. 252, no 3, 204-212 p.
National Category
Engineering and Technology
URN: urn:nbn:se:uu:diva-95862DOI: 10.1016/j.ijms.2005.10.009OAI: oai:DiVA.org:uu-95862DiVA: diva2:170224
Available from: 2007-05-07 Created: 2007-05-07 Last updated: 2016-06-22Bibliographically approved
In thesis
1. Secondary and Higher Order Structural Characterization of Peptides and Proteins by Mass Spectrometry
Open this publication in new window or tab >>Secondary and Higher Order Structural Characterization of Peptides and Proteins by Mass Spectrometry
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work in this thesis has demonstrated the advantages and limitations of using MS based technologies in protein and peptide structural studies.

Tandem MS, specifically electron capture dissociation (ECD) have shown the ability to provide structural insights in molecules containing the slightest of all modifications (D-AA substitution). Additionally, it can be concluded that charge localization in molecular ions is best identified with ECD and to a lesser degree using CAD.

Fragment ion abundances are a quantifiable tool providing chiral recognition (RChiral). An analytical model demonstrating the detection and quantification of D-AAs within proteins and peptides has been achieved. ECD has demonstrated the ability to quantify stereoisomeric mixtures to as little as 1%. Chirality elucidation on a nano LC-MS/MS time scale has been shown.

The structures of various stereoisomers of the mini protein Trp Cage were explored, each providing unique ECD fragment ion abundances suggestive of gas phase structural differences. The uniqueness of these abundances combined with MDS data have been used in proposing a new mechanism in c and z fragment ion formation in ECD. This mechanism suggests initial electron capture on a backbone amide involved in (neutral) hydrogen bonding.

The wealth of solution phase (circular dichroism), transitition phase (charge state distribution, CSD) and gas phase (ECD) data for Trp Cage suggest that at low charge states (2+) the molecule has a high degree of structural similarity in solution- and gas- phases. Furthermore, quantitative information from CSD studies is garnered when using a “native” deuteriated form as part of the stereoisomeric mixture. It has also been shown that the stability of the reduced species after electron capture is indicative of the recombination energy release, which in turn is linked to the coulombic repulsion- a structural constraint that can be used for approximation of the inter-charge distance for various stereoisomers.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. 58 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 304
Engineering physics, Protein Structure, Mass Spectrometry, Electron Capture Dissociation, Tandem Mass Spectrometry, Teknisk fysik
urn:nbn:se:uu:diva-7861 (URN)978-91-554-6887-3 (ISBN)
Public defence
2007-05-28, B41, BMC, Husarg. 3, Uppsala, 10:15
Available from: 2007-05-07 Created: 2007-05-07Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text
By organisation
MMS, Medical Mass SpectrometryIon Physics
In the same journal
International Journal of Mass Spectrometry
Engineering and Technology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 241 hits
ReferencesLink to record
Permanent link

Direct link