Milad Nazari (Presenter)
North Carolina State University
Bio: I am a second-year graduate student in Prof. Muddiman’s group at North Carolina State University, where I am working toward obtaining my PhD in chemistry. In 2013, I graduated from North Carolina State University, where I earned a BS in chemistry (Magna Cum Laude). In my senior year I worked as an undergraduate research assistant where I helped develop novel separation methods of membrane proteins using coacervates. Currently, I’m involved in different projects utilizing mass spectrometry to tackle bioanalytical challenges. My main research project revolves around investigating the aberrant patterns of the lipidome in ovarian cancer. I plan to approach this project using mass spectrometry imaging (MSI) and shotgun lipidomics, where the spatial distribution and relative abundance of different lipids can be investigated using MSI and lipids of interest can then be absolutely quantified usi
Authorship: Milad Nazari, David C. Muddiman
North Carolina State University
| Short Abstract Mass spectrometry imaging (MSI) is a rapidly evolving field for monitoring the spatial distribution and abundance of analytes in biological tissue sections. It allows for direct and simultaneous analysis of hundreds of different compounds in a label-free manner. In order to obtain a comprehensive metabolite and lipid data, a polarity switching MSI method using infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was developed and optimized where the electrospray polarity was alternated from one voxel to the next. Healthy and cancerous ovarian hen tissue sections were analyzed using this method. Distribution and relative abundance of different metabolites and lipids within each tissue section were discerned, and differences between the two were revealed. |
Long Abstract
Introductions:
Lipid profiles provide valuable information for understanding the biological basis of disease. Most lipid classes consist of a polar headgroup and a non-polar hydrocarbon tail. Differences in headgroup composition lead to significant differences in ionization yield between classes. Generally, lipids that are detected in high abundance as positive ions are poorly detected or not detected at all as negative ions, and vice-versa. Therefore, more comprehensive lipid data can be obtained by analyzing the tissue sections in both polarities.
Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization source that has shown significant promise in analysis of biological tissues. The distribution of different lipid classes in chicken ovary tissues was elucidated using IR-MALDESI operated in polarity switching mode.
Methods:
Ovarian tissues were obtained from age-matched healthy and cancerous hens. Tissues were sectioned into 10-µm thick sections at -20 °C. The tissue sections were thaw-mounted onto pre-cleaned glass microscope slides, and were analyzed right away.
In IR-MALDESI analyses, a thin layer of ice is formed over the tissue as the energy-absorbing matrix. A mid-IR laser operated at a wavelength of 2940 nm is then used to resonantly excite the O–H stretching mode of water molecules present in biological tissue and exogenous ice layer, facilitating the desorption of neutral molecules from the tissue. The desorbed material partitions into charged droplets of an orthogonal electrospray plume and ions are generated by an ESI-like process that are sampled by the mass spectrometer. The IR-MALDESI imaging source was fully synchronized with a Thermo Fisher Scientific Q-Exactive Plus mass spectrometer in order to achieve high mass accuracy in lower ppm range. Different modifiers at various concentrations (20 mM ammonium hydroxide, 5 mM ammonium acetate, 1 mM acetic acid, and 100 mM TFE) were added to a 50/50 (v/v) solution of methanol/water in order to optimize the electrospray solvent for polarity switching MSI.
Two laser pulses (f = 20 Hz) per voxel were used to facilitate complete and reproducible desorption of material from the tissue. Ions generated by both laser pulses were accumulated into the C-trap for a fixed injection time (IT = 110 ms), and measured in a single Orbitrap acquisition. An instrument acquisition method was created using the accompanied software to acquire full MS spectra with alternating polarities at each voxel (Fig. 1A). A delay was incorporated into the method using the IR-MALDESI control software, where the signal for laser desorption and the subsequent signal acquisition by MS was delayed by 96 ms in order to allow the electrospray to stabilize upon polarity switching. The desorption diameter (spot size) on tissue was measured to be 150 μm. Spatial resolution of 100 μm was achieved by using the over- sampling method. The images were generated using MSiReader, a free and open-source soft- ware developed for processing high resolving power MSI data, by selecting the desired ionization mode (positive or negative). All images were generated using a bin width of 5 ppm, without any normalization or interpolation.
Results:
Preliminary data collected in both positive- and negative-ion modes revealed different ionization efficiencies for lipids in mouse liver tissue. Two serial sections of mouse liver tissue were imaged using each ionization mode. In general, phosphatidylcholines (PC) and sphingomyelins (SM) exhibited significantly higher ionization efficiencies in positive-ion mode, whereas phosphatidylethanolamines (PE) and phosphatidylinositols (PI) were ionized more efficiently in negative-ion mode.
Mass spectrometry imaging using polarity switching affords the same information, while shortening the data acquisition time by half and allowing all measurements to be made from the same tissue section. However, a potential drawback or limitation of polarity switching is that the electrospray solvent composition would be optimized for one polarity or the other, which could favor one ionization mode over the other. In order to ensure comprehensive coverage of metabolites in both ionization modes, the electrospray solvent composition was optimized for polarity switching MSI. A mouse liver tissue was used to investigate the effects of adding different modifiers such as weak acids, salts and other bases to the electrospray solvent. The effects of these modifiers on image quality, average ion abundance, and %RSD per voxel were investigated, and an optimized condition was identified.
Using the optimized electrospray solvent composition, the polarity switching method was then used to analyze hen ovary tissues (control versus cancerous) in order to investigate the distribution of complex lipids and their possible roles in development of ovarian cancer. Using this method, differences in spatial distribution and relative abundance of hundreds of metabolites (399 in control, and 727 in cancer) was readily discerned. A higher number of fatty acids, phospholipids, sterols, and sphingolipids were identified in the cancerous tissue section. These results were in good agreement with those obtained using conventional methods such as LC-MS/MS and even NMR. This high degree of agreement confirms the utility of polarity switching IR-MALDESI MSI for screening changes in metabolism in cancer cells by simultaneously monitoring the spatial distribution and relative abundances of hundreds of metabolites and lipids.
Conclusions:
This work demonstrates the development of a polarity switching IR-MALDESI MSI method for screening the spatial distribution and relative abundance of different metabolites and lipids in healthy and cancerous tissues. The electrospray solvent was optimized for these analyses in order to obtain a comprehensive metabolite and lipid data, and fundamental mass spectrometry concepts were used to identify the analytes after database searching. Lipid classes such as fatty acids, phospholipids, and sphingolipids exhibited significantly higher relative abundances in cancerous tissue compared to the healthy tissue.
References & Acknowledgements:
We would like to thank Prof. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences and Prof. James Petitte from NCSU Department of Poultry Science for providing the mouse liver and hen ovary tissues, respectively. The authors also gratefully acknowledge financial assistance received from the National Institutes of Health (R01GM087964), the W. M. Keck foundation, and North Carolina State University.
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