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Nanoparticles and photochemistry for Native-like transmembrane protein footprinting

ABSTRACT: Mass spectrometry based footprinting can probe higher order structure of proteins. We bond photocatalytic nanoparticles to a lipid bilayer that, upon laser irradiation, produce high local concentrations of radicals that penetrate the lipid layer made permeable by a simultaneous laser-initiated PB reaction. This approach achieves high footprinting coverage for membrane proteins in liposomes, helps locate both ligand-binding residues in a transporter and ligand-induced conformational changes, and reveals structural aspects of the flexible unbound state. Overall, this approach proves highly effective in intramembrane footprinting and forges a connection between materials science and biology.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Jie Sun

LAB HEAD: Michael L. Gross

PROVIDER: PXD027819 | Pride | 2021-09-26

REPOSITORIES: Pride

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Nanoparticles and photochemistry for native-like transmembrane protein footprinting.

Sun Jie J Liu Xiaoran Roger XR Li Shuang S He Peng P Li Weikai W Gross Michael L ML

Nature communications 20211214 1

Mass spectrometry-based footprinting can probe higher order structure of soluble proteins in their native states and serve as a complement to high-resolution approaches. Traditional footprinting approaches, however, are hampered for integral membrane proteins because their transmembrane regions are not accessible to solvent, and they contain hydrophobic residues that are generally unreactive with most chemical reagents. To address this limitation, we bond photocatalytic titanium dioxide (TiO<sub ...[more]

PMID: 34907205

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Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.

			Action	DRS
	checksum.txt	Txt
	fasta-3kp9.C121_Chymo.txt	Txt
	fasta-hGLUT1.txt	Txt
	hglut1-TiO2-2.raw	Raw
	hglut1-TiO2.csv	Csv