Fragmentation of Integral Membrane Proteins in the Gas Phase
Open accessAbstract
Integral membrane proteins (IMPs) are of great biophysical and clinical interest because of the key role they play in many cellular processes. Here, a comprehensive top down study of 152 IMPs and 277 soluble proteins from human H1299 cells including 11 087 fragments obtained from collisionally activated dissociation (CAD), 6452 from higher-energy collisional dissociation (HCD), and 2981 from electron transfer dissociation (ETD) shows their great utility and complementarity for the identification and characterization of IMPs. A central finding is that ETD is ∼2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed ∼4-fold bias toward CAD and HCD. The location of charges just prior to dissociation is consistent with this directed fragmentation: protons remain localized on basic residues during ETD but easily mobilize along the backbone during collisional activation. The fragmentation driven by these protons, which is most often observed in transmembrane domains, both is of higher yield and occurs over a greater number of backbone cleavage sites. Further, while threshold dissociation events in transmembrane domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from the nearest charge site (R, K, H, N-terminus), fragmentation is strongly influenced by the N- or C-terminal position relative to that site: the ratio of observed b- to y-fragments is ∼1:3 if the cleavage occurs >7 residues N-terminal and ∼3:1 if it occurs >7 residues C-terminal to the nearest basic site. Threshold dissociation products driven by a mobilized proton appear to be strongly dependent on not only relative position of a charge site but also N- or C-terminal directionality of proton movement.
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Earlier top-down LC-MS of denatured integral membrane protein monomers found that transmembrane domains were more likely to fragment in collisional than electron-based dissociation.
“This was consistent with earlier work by Kelleher and co-workers, who performed top-down LC-MS of denatured integral membrane protein monomers, and found that transmembrane domains were more likely to fragment in collisional than electron-based dissociation.111”
From Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry (2020) · cites this paper as [111]
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- Authors
- Skinner, O. S., Catherman, A. D., Early, B. P., Thomas, P. M., Compton, P. D., & Kelleher, N. L.
- Affiliations
- Northwestern University (US)
- Corresponding author
- Kelleher, Neil L.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2014-04-01
- Volume
- 86
- Issue
- 9
- Pages
- 4627-4634
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC4018139
- OpenAlex
- W2092597985
- Cited by
- 29
- References
- 52
- Citations by year
- 2025: 2 · 2024: 4 · 2023: 1 · 2021: 2 · 2020: 4 · 2019: 1 · 2018: 2 · 2017: 4 · 2015: 6 · 2014: 3
- FWCI
- 1.86
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Analytical Chemistry (publishedVersion, other-oa)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National institute of Health and Medical Research (R01 GM067193)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- integral membrane proteins, gas-phase fragmentation, electron transfer dissociation, collisional activation, higher-energy collisional dissociation, transmembrane domain, charge localization, proton mobility, top-down proteomics, H1299 cells, protein identification
- MeSH terms
- Amino Acid Sequence, Gases, Membrane Proteins, Molecular Sequence Data
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