Several studies link mental health problems such as major depression, bipolar disorder and schizophrenia with G-protein coupled receptors (GPCRs), including adrenergic, dopaminergic and cholinergic receptors. Simultaneously, there are more than 140 GPCRs identified through the human genome project where their endogenous ligand is still unknown. In search of these ligands, there is an emerging need for methods that can provide detailed information of ligand affinity and their putative cellular response. Investigations of ligand? receptor interactions should ideally take place as close as possible to the native state of a biological cell with minimal perturbation. Here, we propose to develop a novel probe of cell function, based on ambient sampling mass spectrometry (MS), where ligand?receptor interactions can be monitored in situ by forming a liquid junction between two fused silica capillaries and immersing them on top of a surface with cells acting as a biological model for neurotransmission. The first capillary acts as a perfusion system, the second capillary pulls on the liquid junction and delivers a spray of analytes at the MS-inlet for detection. The capability of measuring a ligand-receptor binding event, the subsequent release of neurotransmitters and any enzymatic conversion of the released molecules taking place on the cell surface is made possible by the multiplexing abilities of MS. The technique does not require derivatization to detect analytes and is non-invasive. Using the PC12 cell line, the affinity of several classic neurotransmitters will be screened against endogenous muscarinic receptors as a proof-of-concept for this novel method. The technique will also be used to perform measurements of cellular responses such as release of dopamine from PC12 cells. The establishment of the proposed technique is highly translational in the sense that it may be applied to virtually any type of tissue and ligand-receptor interaction. Understanding the complexity of cellular mechanisms in neurons is a challenging task as recognized by the BRAIN initiative and ligand-receptor interactions are a fundamental part of cell-to-cell signaling. The proposed groundbreaking research will develop a method for measuring ligand?receptor interactions on the surface of a cell using mass spectrometry. To detect and measure these events with ambient sampling coupled to MS has hitherto never been done. This is a challenging yet possible task that will be achieved through collaboration between the Zare group at Stanford University and the Zhong group at Central China Normal University. The project combines recent advances of ambient sonic spray ionization with classic microperfusion, nanoflow liquid chromatography and high-resolution MS, and is expected to interrogate ligand?receptor interactions on live cells.

Public Health Relevance

We propose to develop a novel probe of cell function, where a laminar flow bridge based on fused silica capillaries interrogates ligand?receptor interactions directly on live cells and is analyzed with ambient ionization mass spectrometry in real time. This research is relevant to public health for characterizing and understanding receptor mechanisms induced by the binding event of neurotransmitters and neuropeptides. Such information obtained with mass spectrometry is expected to give highly detailed chemical information about the effects that nervous disorders have on cellular functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH112188-03
Application #
9600078
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Friedman, Fred K
Project Start
2017-01-01
Project End
2019-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Jansson, Erik T; Lai, Yin-Hung; Santiago, Juan G et al. (2017) Rapid Hydrogen-Deuterium Exchange in Liquid Droplets. J Am Chem Soc 139:6851-6854
Dulay, Maria T; Zare, Richard N (2017) Polymer-spray mass spectrometric detection and quantitation of hydrophilic compounds and some narcotics. Rapid Commun Mass Spectrom 31:1651-1658
Hsu, Cheng-Chih; ElNaggar, Mariam S; Peng, Yao et al. (2013) Real-time metabolomics on living microorganisms using ambient electrospray ionization flow-probe. Anal Chem 85:7014-8
Olesen, Kenneth; Karlsson, Roger; Lind, Ulrika et al. (2013) Detection of ligand-receptor binding using microfluidic frontal affinity chromatography on proteoliposomes derived directly from native cell membranes. J Chromatogr B Analyt Technol Biomed Life Sci 931:84-9
Santos, Vanessa G; Regiani, ThaĆ­s; Dias, Fernanda F G et al. (2011) Venturi easy ambient sonic-spray ionization. Anal Chem 83:1375-80