Development of enabling technologies for single cell analysis with mass spectrometry

Mass spectrometry (MS) is an effective methodology for untargeted, label-free, highly multiplexed analyses of trace compounds based on their mass-to-charge ratios. For biological applications, these properties have generated interest in determining biomarkers of diseased states, detecting drug compounds and metabolites, and observing previously unknown chemical messengers. Recent developments in instrumentation have provided exquisite sensitivity with robust performance. A growing field of single cell chemical analysis has arisen around these figures of merit. While early reports utilized manual isolation and extraction, recent developments in high-throughput sampling have enabled the examination of large populations of cells. One such method includes the analysis of dispersed single cells on a flat surface. When cells are randomly seeded onto the surface, their locations have to be determined by optical imaging to direct acquisition of isolated cells efficiently. A variety of microprobe ionization sources are suitable for such analyses, though smaller probe footprints can utilize more densely seeded samples. This dissertation describes two technologies for performing single cell analysis with mass spectrometry. The first, synchronized desorption electrospray ionization (DESI), facilitates ambient ionization MS with high mass resolution, low duty cycle mass analyzers. The initial report utilized synchronized DESI for mass spectrometry imaging, but interrupting the desorption plume would be useful for profiling several locations on a surface in an arbitrary order for single cell analysis. The second methodology utilizes microscopy images to guide MS profiling. Specifically, image analysis software, called microMS, was developed to perform cell finding and correlate optical coordinates with the physical coordinates in a mass spectrometer. Since most of the functionality of microMS is decoupled from the mass spectrometer, the workflow can be easily extended to a variety of instruments. Using matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)-MS, rodent pancreatic islet cells were investigated and heterogeneous peptide processing was detected at the single cell level. With secondary ion mass spectrometry, disparate tissue from the mammalian nervous system was differentiated and further stratified into separate populations. A unique feature of such analyses is that only a fraction of the sample is consumed and the location of a cell is constant once the sample is dried. This property greatly simplifies sequential, follow-up analysis. As an example, MALDI-TOF-MS was utilized to rapidly screen a population of islet cells to select alpha and beta cell types. The locations of those cells were then targeted for liquid microjunction extraction in order to examine their metabolite profiles with capillary electrophoresis-MS. Finally, while microscopy-guided MS profiling is accurate enough to target single cells, the methodology is flexible enough to analyze much larger samples, including tissue sections or bacterial colonies. As an application, natural product mutant libraries were screened directly from E. coli colonies using microMS. The suite of technologies and protocols described increases the applicability of many mass spectrometers to characterize a range of cells, colonies and similar objects for their chemical composition

Porous Phospholipid Nanoshells as Enzymes Delivery Agents

Diabetes is an epidemic in developed nations. Glucokinase (GK) is vital for glocose sensing, and is directly implicated in particular forms of diabetes. Studying pancreatic cells with altered GK activity would facilitate studies, but current methods for altering proteomes are lacking. Porous phospholipid nanoshells (PPNs) have traditionally been used as platforms for biologically derived nanosensors, though their biocompatibility and protease resistance well suits them as enzyme delivery agents. GK kinetics were investigated with an enzyme coupled reaction to determine the effect of encapsulation. It was determined that encapsulation increased the Hill coefficient by 5.8% and the S(0.5) by 1.8%. This small deviation may not be significant in physiological conditions. To observe a recovered function in cell lines upon reintroducing GK, constitutively expressed GK must first be knocked down with siRNA. As initial work toward an siRNA knockdown, immunoblotting conditions were optimized resulting in a detection limit below 10 ng of GK. Immunoblotting verified suspected constitutive expression of GK in INS-1 cell lines. While further investigation is necessary to demonstrate the utility of GK-containing PPNs for cell delivery, this thesis outlines the generation and characterization of this novel enzyme delivery platform

Pulsed Desorption Electrospray Ionization Mass Spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful method for generating ions at ambient conditions without the need for sample preparation or pretreatment. In DESI-MS, a continuous stream of charged microdroplets impacts a surface containing a sample (cDESI-MS). Upon impact, analyte molecules are extracted from the surface into secondary microdroplets, from which gas-phase ions are eventually formed. We have developed a pulsed DESI-MS source (pDESI-MS) that demonstrates higher sensitivity. In addition, the pDESI-MS source is designed so that desorption only occurs during ion accumulation time (IT) of an ion trap mass spectrometer, increasing the sampling efficiency (SE) closer to 100%. This capability is particularly advantageous for high-resolution instruments such as the LTQOrbitrap hybrid mass spectrometers that require significant transient acquisition times after ion accumulation (e.g. for IT = 0.5 s and m/z-resolution = 100,000, and scan time = ~2.3 s, the SE increases by a factor of ~5 from 22% to 100%). In addition, pulsing the primary microdroplet spray reduces the total volume of deposited solvent per m/z scan, which minimizes the ‘washing effect’ reported for cDESI-MS on surfaces such as glass or Teflon®. The ability to control the amount of deposited solvent by varying the pulse widths offer the potential for improved spatial resolution in imaging mode. These results demonstrate that pDESI-MS offers the potential to transform current approaches for implementing spray-based ambient ionization techniques in chemical analysis and imaging

Pulsed Desorption Electrospray Ionization Mass Spectrometry Imaging

Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful method for generating ions at ambient conditions without the need for sample preparation or pretreatment. In DESI-MS, a continuous stream of charged microdroplets impacts a surface containing a sample (cDESI-MS). Upon impact, analyte molecules are extracted from the surface into secondary microdroplets, from which gas-phase ions are eventually formed. We have developed a pulsed DESI-MS source (pDESI-MS) that demonstrates higher sensitivity. In addition, the pDESI-MS source is designed so that desorption only occurs during ion accumulation time (IT) of an ion trap mass spectrometer, increasing the sampling efficiency (SE) closer to 100%. This capability is particularly advantageous for high-resolution instruments such as the LTQOrbitrap hybrid mass spectrometers that require significant transient acquisition times after ion accumulation (e.g. for IT = 0.5 s and m/z-resolution = 100,000, and scan time = ~2.3 s, the SE increases by a factor of ~5 from 22% to 100%). In addition, pulsing the primary microdroplet spray reduces the total volume of deposited solvent per m/z scan, which minimizes the ‘washing effect’ reported for cDESI-MS on surfaces such as glass or Teflon®. The ability to control the amount of deposited solvent by varying the pulse widths offer the potential for improved spatial resolution in imaging mode. These results demonstrate that pDESI-MS offers the potential to transform current approaches for implementing spray-based ambient ionization techniques in chemical analysis and imaging

Label-free assay for classifying intact human breast cancer cells using desorption electrospray ionization mass spectrometry

Endogenous fatty acid synthesis is up-regulated in breast cancer. Phospholipids have been implicated in a number of cellular processes including signaling, proliferation, and apoptosis, but their role in cancer is largely unknown. In an effort to understand how lipids mediate tumor progression and oncogene expression, intact human various breast cancer cells having were interrogated by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). This in vitro cell-based workflow provides rapid and detailed analysis with little sample preparation compared to traditional lipid separation and extraction methods. Principal component analyses of cell lipid profiles allowed identification of cell lines based on differential expression of both Her2 and p53 genes, differences in the effect of these mutations on cancer progression. Breast cancer cell lines representing changes in metastatic potential and grade were also easily distinguished within a single assay. Tandem MS analyses identified of over 200 individual lipid species in negative ion mode, which is currently the most comprehensive phospholipid composition of breast cancer cells. These results demonstrate that DESI-MS based assays are powerful tools for rapidly classifying of cancer cells according to their geno- and phenotypes, while simultaneously providing extensive lipid compositions relevant to elucidating important biochemical mechanisms in cancer progression

Pulsed Desorption Electrospray Ionization Mass Spectrometry Imaging

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a powerful method for generating chemical maps of surfaces and tissues at ambient conditions without the need for sample preparation. In DESI-MSI, a continuous stream of microdroplets impacts a sample affixed to a XY translation stage (cDESI-MSI). Upon impact, analyte molecules are extracted from the surface into secondary microdroplets, from which gas-phase ions are eventually formed. We have developed a pulsed DESI-MSI source (pDESI-MSI) that demonstrates approx. 5-10 times improvement in sensitivity compared to cDESI-MSI. The pDESI-MSI source is designed so that desorption only occurs during the ion accumulation time (IT) of an ion trap mass spectrometer, which increases sampling efficiency (SE) close to 100%. In addition, pulsing the primary microdroplet spray reduces the total volume of deposited solvent per pixel, minimizing the \u27washing effect\u27 and improving spatial resolution. This capability is particularly advantageous for coupling DESI-MSI to high-resolution ion trap instruments that have lengthy transient acquisition times (e.g. LTQ-Orbitrap XL: IT = 0.5 s, m/z-resolution = 100,000, scan time = approx. 2.30 s, pDESI-MSI increases SE from approx. 22% to 100%). pDESI-MSI offers the potential to acquire multiple DESI-MSI scan types per pixel without degrading spatial resolution, thereby providing more chemical information and better spatial accuracy of co-registered images. Implementation of pDESI-MSI also suggests a potential avenue for improving the performance of other continuous spray-based ambient ionization MSI techniques

Multistage Reactive Transmission-Mode Desorption Electrospray Ionization Mass Spectrometry

Elucidating reaction mechanisms is important for advancing many areas of science such as catalyst development. It is often difficult to probe fast reactions at ambient conditions with high temporal resolution. In addition, systems involving reagents that cross-react require analytical methods that can minimize interaction time and specify their order of introduction into the reacting system. Here, we explore the utility of transmission mode desorption electrospray ionization (TM-DESI) for reaction monitoring by directing a microdroplet spray towards a series of meshes with micrometer-sized openings coated with reagents, an approach we call multistage reactive TM-DESI (TM n -DESI, where n refers to the number of meshes; n = 2 in this report). Various stages of the reaction are initiated at each mesh surface, generating intermediates and products in microdroplet reaction vessels traveling towards the mass spectrometer. Using this method, we investigated the reactivity of iron porphyrin catalytic hydroxylation of propranolol and other substrates. Our experimental results indicate that TM n-DESI provides the ability to spatially separate reagents and control their order of introduction into the reacting system, thereby minimizing unwanted reactions that lead to catalyst deactivation and degradation products. In addition, comparison with DESI-MS analyses (the Zare and Latour laboratories published results suggesting accessible reaction times \u3c1 ms) of the reduction of dichlorophenolindophenol by L-ascorbic acid suggest that TM 1 -DESI can access reaction times less than 1 ms. Multiple meshes allow sequential stages of desorption/ionization per MS scan, increasing the number of analytes and reactions that can be characterized in a single experiment

Pulsed Desorption Electrospray Ionization Mass Spectrometry Imaging

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a powerful method for generating chemical maps of surfaces and tissues at ambient conditions without the need for sample preparation. In DESI-MSI, a continuous stream of microdroplets impacts a sample affixed to a XY translation stage (cDESI-MSI). Upon impact, analyte molecules are extracted from the surface into secondary microdroplets, from which gas-phase ions are eventually formed. We have developed a pulsed DESI-MSI source (pDESI-MSI) that demonstrates approx. 5-10 times improvement in sensitivity compared to cDESI-MSI. The pDESI-MSI source is designed so that desorption only occurs during the ion accumulation time (IT) of an ion trap mass spectrometer, which increases sampling efficiency (SE) close to 100%. In addition, pulsing the primary microdroplet spray reduces the total volume of deposited solvent per pixel, minimizing the \u27washing effect\u27 and improving spatial resolution. This capability is particularly advantageous for coupling DESI-MSI to high-resolution ion trap instruments that have lengthy transient acquisition times (e.g. LTQ-Orbitrap XL: IT = 0.5 s, m/z-resolution = 100,000, scan time = approx. 2.30 s, pDESI-MSI increases SE from approx. 22% to 100%). pDESI-MSI offers the potential to acquire multiple DESI-MSI scan types per pixel without degrading spatial resolution, thereby providing more chemical information and better spatial accuracy of co-registered images. Implementation of pDESI-MSI also suggests a potential avenue for improving the performance of other continuous spray-based ambient ionization MSI techniques

Development of Multistage Reactive Transmission Mode Desorption Electrospray Ionization Mass Spectrometry for characterizing complex catalytic reactions

Characterizing transient solution-phase intermediates of catalytic reactions at ambient conditions is a long-standing problem in analytical chemistry. Electrospray ionization mass spectrometry (ESI-MS) is well-established for studying reaction mechanisms in solution. Stopped- and continuous-flow ESI-MS can achieve time resolution in the millisecond (ms) regime (as low as ∼5-10 ms) but their complicated experimental configurations limit throughput and the scope of reactions available for study. In addition, these methods are prone to carryover effects, which can cause unwanted side reactions during analysis. Ambient mass spectrometry (MS) techniques such as reactive desorption electrospray ionization (rDESI) has resolved many of these problems, achieving reaction time resolution in the millisecond regime. Herein, we have successfully developed an ionization source based on transmission mode DESI (TM-DESI) called multi-stage reactive TM-DESI (rTMn-DESI, where n represents the number of desorption stages) that allows real-time capture of intermediates on submillisecond timescales. In addition, this technique allows control of the order in which reagents are introduced, providing the ability for step-wise elucidation of fast catalytic solution-phase processes and for minimizing unwanted degradation reactions. Using this technique, we characterize intermediates of various catalytic reactions such as C-H hydroxylation via high-valent iron oxo porphyrin complexes