Mapping Peptide Thiol Accessibility in Membranes Using a Quaternary Ammonium Isotope-Coded Mass Tag (ICMT)
Abstract
The plasma membrane contains a diverse array of proteins, including receptors, channels, and signaling complexes, that serve as decision-making centers. Investigation of membrane protein topology is important for understanding the function of these types of protein. Here, we report a method to determine protein topology in the membrane that utilizes labeling of cysteine with isotope-coded mass tags. The mass tags contain a thiol reactive moiety, linker, and a quaternary ammonium group to aid ionization in the mass spectrometer and were synthesized in both light and heavy (deuterated) forms. The probes were found to be membrane impermeable when applied to lipid vesicles. To assess the utility of the probes for mapping peptide thiol topology, we employed a two-step labeling procedure. Vesicles containing α-helical transmembrane peptides were labeled with heavy (or light) probe, solubilized by detergent, and then labeled by an excess of the complementary probe. Peptide for which the cysteine was oriented in the center of the lipid bilayer was not labeled until the lipid vesicles were lysed with detergent, consistent with the membrane impermeability of the probes and reduced ionization of the thiol in the hydrophobic membrane. Peptide for which the cysteine was positioned in the headgroup zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the headgroup abutting the hydrocarbon region was labeled at a reduced rate compared to the fully accessible cysteine. Moreover, the effect of lipid bilayer structure on the kinetics of peptide and lipid flipping in the bilayer was readily measured with our two-step labeling method. The small sample size required, the ease and rapidity of sample preparation, and the amenability of MALDI-TOF mass spectral analysis to the presence of lipids will enable future facile investigation of membrane proteins in a cellular context.
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This article is cited by 5 publications.
- John E. Gadbery, Nicole S. Sampson. Use of an Isotope-Coded Mass Tag (ICMT) Method To Determine the Orientation of Cholesterol Oxidase on Model Membranes. Biochemistry 2018, 57 (36) , 5370-5378. https://doi.org/10.1021/acs.biochem.8b00788
- Jamie LeBarron and Erwin London . Highly Hydrophilic Segments Attached to Hydrophobic Peptides Translocate Rapidly across Membranes. Langmuir 2016, 32 (41) , 10752-10760. https://doi.org/10.1021/acs.langmuir.6b02597
- Megan H. Touchette, Gopal R. Bommineni, Richard J. Delle Bovi, John E. Gadbery, Carrie D. Nicora, Anil K. Shukla, Jennifer E. Kyle, Thomas O. Metz, Dwight W. Martin, Nicole S. Sampson, W. Todd Miller, Peter J. Tonge, and Jessica C. Seeliger . Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl β-Diol Lipids. Biochemistry 2015, 54 (35) , 5457-5468. https://doi.org/10.1021/acs.biochem.5b00455
- John E. Gadbery, Nicole S. Sampson. A mass spectrometry-based isotope-coded mass tag method to map thiol accessibility in biological systems. 2019, 245-260. https://doi.org/10.1016/bs.mie.2019.02.025
- Fuzhong Ouyang, Zhihao Zhao, Ruifang Gao, Rui Shi, Enhui Wu, Rui Lv, Guoqiang Xu, Jian Liu. Dual Maleimide Tagging for Relative and Absolute Quantitation of Cysteine‐Containing Peptides by MALDI‐TOF MS. ChemBioChem 2018, 19 (11) , 1154-1161. https://doi.org/10.1002/cbic.201800031