Concurrent Scientific Session (Imaging and Mass Spectrometry): Imaging and Spatially Explicit Mass Spectrometry
Desorption Electrospray Ionization – Mass Spectrometry Imaging: Recent Developments and Perspectives
Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is an ambient ionization MS method utilized in over 730 peer-reviewed manuscripts. In this technique, a highly charged aerosol of microdroplets is sprayed onto a surface, desorbing and ionizing molecules which are then analyzed by MS. Since samples are analyzed in their native conditions with minimal to no sample preparation (e.g. without the need for separation or an organic matrix), chemicals, drugs, metabolites and lipids are rapidly detected and mapped in diverse types of samples. We focus on three recent DESI-MSI developments: (1) intraoperative brain cancer diagnostics, (2) developmental biology, and (3) high-throughput chemical reaction screening. For intraoperative brain cancer diagnostics, DESI-MS is being used in operating rooms for rapid tissue biopsy smear analysis to screen for diagnostic lipids and oncometabolites indicative of tumor type (e.g. glioma), grade and extent of tumor infiltration at surgical margins. Recently, isocitrate dehydrogenase (IDH) mutation status was determined intraoperatively by DESI in gliomas, offering new tumor management options which may impact extent of resection goals. In developmental biology, DESI-MSI was applied to whole body 2D and 3D swine fetus cryosections, and a range of lipids and metabolites specific to particular organs were spatially mapped and related to organogenesis. Finally, an autonomous system for high-throughput chemical reaction screening by DESI-MSI is an ongoing effort funded by the Defense Advanced Research Projects Agency (DARPA). This system takes advantage of the fact that chemical reactions are accelerated in DESI microdroplets to read over 1,000 unique reaction spots/hour and to explore the high dimensional chemical reaction space. To accomplish that, DESI-MS is integrated with four other commercial instruments all under computer control (a robotic pipetting robot, a robotic arm, a plate hoteling system and a precision solvent delivery system). In addition, future directions and perspectives for DESI-MSI will be presented.
Single cell proteome mapping of tissue heterogeneity using microfluidic nanodroplet sample processing and ultrasensitive LC-MS
Biological tissues are highly heterogeneous, consisting of a variety of cell types, subpopulations, and substructures. Understanding heterogeneity at the single cell level is of great interest for biomedical research. While MS-based proteomic analyses are capable of quantifying thousands of proteins, the extension to single cell studies has been largely ineffective. This is primarily due to the large sample losses incurred during sample preparation procedures. We have developed nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples), which significantly minimize sample losses during proteomic preparation. nanoPOTS utilizes a robotic platform to dispense nanoliter volumes of reagents into photolithographically patterned nanowell reaction vessels. Sample preparation utilizes a novel workflow that eliminates the need for multiple reaction vessels and cleanup steps to process cellular tissue into purified tryptic peptides. Single cells can be isolated into nanowells by fluorescence-activated cell sorting (FACS) or laser capture dissection (LCM), processed, and analyzed with low-flow nanoLC Orbitrap mass spectrometry. To date, we have identified >3,000 protein groups from as few as 10 HeLa cells, which is a level of proteome coverage not previously achieved from fewer than thousands of cells, and ~700 proteins have been identified from single mammalian cells. To enable high-resolution tissue mapping, we developed an automated method to couple LCM with nanoPOTS. This approach is capable of quantifying ~2000 proteins in 100-µm tissue voxels. We used this approach to study protein expression difference in single pancreas islets from healthy and Type 1 Diabetes human donors. More recently, a high-resolution mapping method was built to generate protein expression images of mouse uterine tissue sections for blastocyst implantation. The ability to map the proteome with high spatial resolution across tissue regions provides a fundamental way to understand the tissue microenvironment, substructure, and cellular organization from a global proteome perspective.
Mass Cytometry Why Multiplex
When contemplating multiplex studies what can be gained and how should the study be structured? What features are important to the study and ultimately why perform the time consuming, costly, and more challenging experiment? There are multiple ways to answer these questions, but my answers may surprise. Most of my perspective derives from what we have learned from other technologies and how we extrapolated those studies. My goal is to inform and help researchers determine their best options for imaging studies using Mass Cytometry. Ultimately, it may all comes down the correct analytical tool and learning what to look for in the clutter. … just imagine