TY - JOUR
T1 - Footprinting Mass Spectrometry of Membrane Proteins
T2 - Ferroportin Reconstituted in Saposin A Picodiscs
AU - Zhou, Fengbo
AU - Yang, Yihu
AU - Chemuru, Saketh
AU - Cui, Weidong
AU - Liu, Shixuan
AU - Gross, Michael
AU - Li, Weikai
N1 - Funding Information:
We thank Henry Rohrs and Don Rempel for support and Ming Cheng, Ke Li, and Jing Li for helpful discussions. This work is supported by NIGMS R01 GM131008 and the American Heart Association 20CSAOI34710002 (W.L. and M.L.G.). The mass spectrometry resource was supported by NIH NIGMS P41GM103422 and R24GM136766 (M.L.G.).
Publisher Copyright:
©
PY - 2021/8/24
Y1 - 2021/8/24
N2 - Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.
AB - Membrane proteins participate in a broad range of cellular processes and represent more than 60% of drug targets. One approach to their structural analyses is mass spectrometry (MS)-based footprinting including hydrogen/deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and residue-specific chemical modification. Studying membrane proteins usually requires their isolation from the native lipid environment, after which they often become unstable. To overcome this problem, we are pursuing a novel methodology of incorporating membrane proteins into saposin A picodiscs for MS footprinting. We apply different footprinting approaches to a model membrane protein, mouse ferroportin, in picodiscs and achieve high coverage that enables the analysis of the ferroportin structure. FPOP footprinting shows extensive labeling of the extramembrane regions of ferroportin and protection at its transmembrane regions, suggesting that the membrane folding of ferroportin is maintained throughout the labeling process. In contrast, an amphipathic reagent, N-ethylmaleimide (NEM), efficiently labels cysteine residues in both extramembrane and transmembrane regions, thereby affording complementary footprinting coverage. Finally, optimization of sample treatment gives a peptic-map of ferroportin in picodiscs with 92% sequence coverage, setting the stage for HDX. These results, taken together, show that picodiscs are a new platform broadly applicable to mass spectrometry studies of membrane proteins.
UR - http://www.scopus.com/inward/record.url?scp=85114029493&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.1c02325
DO - 10.1021/acs.analchem.1c02325
M3 - Article
C2 - 34383472
AN - SCOPUS:85114029493
VL - 93
SP - 11370
EP - 11378
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 33
ER -