Journal
ANALYTICAL CHEMISTRY
Volume 86, Issue 9, Pages 4627-4634Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac500864w
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Funding
- National Institute of General Medical Sciences of the National Institutes of Health [R01 GM067193]
- UIUC Center for Neuroproteomics on Cell to Cell Signaling [P30 DA018310]
- Chicago Biomedical Consortium
- The Searle Funds at The Chicago Community Trust
- Robert H. Lurie Comprehensive Cancer Center
- ACS Division of Analytical Chemistry
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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 similar to 2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed,similar to 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 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 fragments is similar to 1:3 if the cleavage occurs >7 residues N-terminal and similar to 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. transmembrane domains, both is of
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