Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process

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Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M r ) values, for a total of 689 different values. However, only ϳ10% of these values matched (Ϯ1 Da) the DNA predicted M r values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M r matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective. (J Am Soc Mass Spectrom 2003, 14, 253-261)

Section: Glycoprotein Preparationmentioning

confidence: 99%

Top down characterization of secreted proteins from Mycobacterium tuberculosis by electron capture dissociation mass spectrometry

ElNaggar

Sze

et al. 2003

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“…Electron capture dissociation (ECD), for example, has been shown to achieve effective cleavage of large polypeptides and small proteins [8]. This method induces peptide fragmentation, primarily at N-C␣ bonds, using low-energy electrons [9].…”

mentioning

confidence: 99%

“…For instance, facile neutral loss of phosphoric acid, as commonly observed in CAD spectra of phosphopeptides, is rarely observed using ECD. This has prompted a strong focus on the use of ECD for post-translational modification analysis [14], but ECD has also proven successful more generally in generating peptide and protein identification data [8,9,13,15,16]. The implementation of ECD is however largely restricted to ion cyclotron resonance instruments.…”

mentioning

confidence: 99%

Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy

Hart

Lau

Hao

et al. 2009

The use of electron-transfer dissociation as an alternative peptide ion activation method for generation of protein sequence information is examined here in comparison with the conventional method of choice, collisionally activated dissociation, using a linear ion trapping instrument. Direct comparability between collisionally and electron-transfer-activated product ion data were ensured by employing an activation-switching method during acquisition, sequentially activating precisely the same precursor ion species with each fragmentation method in turn. Sequest (Thermo Fisher Scientific, San Jose, CA) searching of product ion data generated an overlapping yet distinct pool of polypeptide identifications from the products of collisional and electron-transfer-mediated activation products. To provide a highly confident set of protein recognitions, identification data were filtered using parameters that achieved a peptide false discovery rate of 1%, with two or more independent peptide assignments required for each protein. The use of electron transfer dissociation (ETD) has allowed us to identify additional peptides where the quality of product ion data generated by collisionally activated dissociation (CAD) was insufficient to infer peptide sequence. Thus, a combined ETD/CAD approach leads to the recognition of more peptides and proteins than are achieved using peptide analysis by CAD-or ETD-based tandem mass spectrometry alone. (J Am Soc Mass Spectrom 2009, 20, 167-175)

“…An alternative dissociation technique is electron capture dissociation (ECD), where low energy electrons are captured by multiply-protonated species [22,23]. This overall process transforms the ion from an evenelectron closed-shell system to an odd-electron hypervalent system while depositing the energy associated with electron capture into the ion.…”

mentioning

confidence: 99%

SO₂^−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

Chrisman

Pitteri

Hogan

et al. 2005

104

122

Multiply-charged peptide cations comprised of two polypeptide chains (designated A and B) bound via a disulfide linkage have been reacted with SO 2 Ϫ· in an electrodynamic ion trap mass spectrometer. These reactions proceed through both proton transfer (without dissociation) and electron transfer (with and without dissociation). Electron transfer reactions are shown to give rise to cleavage along the peptide backbone, loss of neutral molecules, and cleavage of the cystine bond. Disulfide bond cleavage is the preferred dissociation channel and both Chain A (or B)OS · and Chain A (or B)OSH fragment ions are observed, similar to those observed with electron capture dissociation (ECD) of disulfide-bound peptides. Electron transfer without dissociation produces [M ϩ 2H] ϩ· ions, which appear to be less kinetically stable than the proton transfer [M ϩ H] ϩ product. When subjected to collision-induced dissociation (CID), the [M ϩ 2H] ϩ· ions fragment to give products that were also observed as dissociation products during the electron transfer reaction. However, not all dissociation channels noted in the electron transfer reaction were observed in the CID of the [M ϩ 2H] ϩ· ions. The charge state of the peptide has a significant effect on both the extent of electron transfer dissociation observed and the variety of dissociation products, with higher charge states giving more of each. ( T andem mass spectrometry currently plays a major role in the identification and characterization of proteins [1][2][3]. This application is enabled by the ability to form gaseous ions from peptides and proteins, typically via electrospray ionization [4,5] or matrix-assisted laser desorption ionization [6,7], the ability to form fragments from peptides and proteins of interest that reveal primary structure information, and the ability to measure and detect the fragments. Amino acid sequence information is usually sought for the identification of a protein. However, the identities and locations of post-translational modifications arising from, for example, phosphorylation, glycosylation, and disulfide bonding are also of interest for the complete characterization of the protein of interest. Unimolecular dissociation is the predominant chemical means for deriving primary structure information from a peptide or protein in the gas phase. The extent to which structural information can be derived from a posttranslationally modified peptide or protein depends upon many factors including, for example, charge state and nature of the ion (e.g., protonated versus metal cationized), nature of the modification, and ion activation conditions. The nature of the modification itself can play a major role in directing dissociation chemistry. For this reason, it is of interest to explore various means for both forming and activating post-translationally modified ions. In this study, we focus on the behavior of polypeptide chains bound by a cystine bridge. The formation of such bridges takes place as a protein folds into its native conformation, and the bri...