Mariam Elnaggar (Presenter)
prosolia
Bio: Despite earlier work in hospital pharmacy and contract research environs, Mariam’s primary work with mass spectrometry began as a biologist and chemist at Cornell University, with a proteomic focus, under Fred McLafferty. Focusing on integration with microfluidics and related atmospheric pressure applications, she conducted her PhD research in the labs of Rich Mathies and Evan Williams at UC Berkeley. To develop a deeper understanding of surface sampling, she became a postdoctoral scholar at Oak Ridge National Laboratory in the group of Gary Van Berkel. Given experience there, when Prosolia began developing a commercial liquid junction based surface sampling system, she joined the team and has since worked with applications on microbial, histological, cellular, and analytical systems.
Authorship: Mariam ElNaggar
Prosolia, Indianapolis, IN
| Short Abstract The flowprobe system facilitates atmospheric pressure extractive sampling without additional extensive preparation for spot based targeted profiling as well as arrayed imaging and scanned profiling. Presented here are a selection of emerging applications of the sample introduction technique at the levels of clinical, quantitative, and basic research, biomarker discovery, and drug deposition analysis. The identification and characterization of molecules of interest via direct and continuous in situ microextraction and ionization based surface analysis is shown to be, importantly, possible from histologic and cytological preparations in such a way that the samples are preserved and useful for subsequent orthogonal analysis. |
Long Abstract
— Introduction —
Devices that enable quantitative characterization of chemical distributions facilitate the adoption and broader usage of mass spectrometry in health care environments. Molecular imaging can directly answer questions with respect to clinical outcomes. These questions tend to be restrictions in workflows and applications as diverse as basic research level microbial biomarker exploration and discovery, understanding of mechanisms of action in drug development, and real-time therapeutic and diagnostic measurements of human derived samples. Acquiring these results quickly and with minimized expense at the laboratory level—which in turn leads to patient care improvements and downstream health care cost reductions--is dependent on consistency and efficiency of analytical methodologies.
Commercial technologies have begun to address generating higher resolution data, but ablation and ionization systems can bias sampling to compounds that are particularly thermally insensitive or interactive with applied matrixes. Liquid Microjunction Surface Sampling Probes can bypass some of these biases. Mass spectrometric analysis based on the impingement of fluid onto surfaces for extraction and ionization (sequentially, in tandem, or continuously), as an atmospheric pressure surface sampling workflow, also benefits from the obviated need for extensive sample preparation as is required in MALDI and other chemical imaging methods.
The continuous in situ microextraction provided by the flowprobe system also enables rapid, high sensitivity analysis with few limitations to sample type and versatile utility as regards experimental operation. Applying flowprobe analysis to soluble films, laminates, and analytical surfaces; liver, brain, and lung tissue infected with tuberculosis; as well as microbial or cytological preparations, collected as part of an extant fine needle aspirate based workflow, resulted in effective characterization of drugs, metabolites, and other biologically derived compounds of interest either as chemical images, time resolved intensity profiles, or mass spectra.
— Methods —
For cytological preparations, Breast Cancer Cells (MDA-MB-231) were harvested, resuspended, preliminarily diluted, and quantified at various concentrations via haemocytometry prior to centrifuge based application to slides utilizing cytospin devices prior to microscopy and flowprobe analysis [1]. As part of a secondary workflow, they were afterwards stained and cover-slipped for comparisons with unsampled controls. Flowprobe analysis through disrupted aspiration and stabilization (DAS), as well as through rastered scans, was performed.
UV-irradiated Lung biopsies from a rabbit TB model, at various time points, were acquired via a collaborator and contained advanced-stage tumors consisting of large necrotic cores, surrounding cellular lesion tissue (granulomas) and areas of non-lesion (normal) lung tissue [2]. In some cases, samples had been from systems treated with Levofloxacin in order to run disposition studies. In addition to full slice chemical imaging, spatial and temporal profiling of tissues by Flowprobe analysis was initiated by extracting from various discrete spots inside different TB granuloma compartments from biopsy sections at each time point. These data could be compared to MALDI and LC/MS/MS data taken with serial sections and tissue homogenates by the collaborator.
In some cases, solid standards of species of interest or soluble films and surfaces were dissolved into and with water, methanol, chloroform, acetonitrile, and other acidified solutions in order to determine optimized extraction conditions [3]. Samples were scanned for analysis and replicates, based on various time points or concentrations or application appropriate statistical analysis, were chosen for in situ extractive analysis.
Microbial samples provided by collaborators were sampled both on petri-dishes in situ as well as by spot sampling of picked and smeared colonies [4].
All specimens were analyzed using the flowprobe system mounted onto various Thermo mass spectrometers, inclusive of an LTQ, Orbitrap, and Hybrid Quadrupole Orbitrap.
Data was acquired in full scan mode covering a window of m/z ranges suitable for simultaneous acquisition of potential drug, protein, metabolite, and lipid signals. In some cases, spectra were integrated over the sampling time to ensure comparable positive ion mode spectra for comparative analysis. In some cases, images were acquired using microscopy to monitor the effects of the extraction on and below layers of substrates or samples of interest.
Chemical images were acquired via automated sequential extractions of surfaces where collected data was compiled and stitched together using the Firefly 2.0 software. Data visualization and images extracted from BioMap3.0 and MSI-Reader will be shown.
— Results —
Data were acquired in order to characterize the profiling and imaging capabilities of the flowprobe utilizing various substrates and analytes and establish limits to imaging analysis. Evaluation of the mass spectral data as well as subsequent data from cells on cytospin slides revealed that fixation and analysis of the samples could be performed such that cells were preserved for subsequent morphological evaluation, even with spectra indicating the quantitative extraction of various phospholipids.
Tissue samples with endogenous compounds of interest and various biopharmaceuticals were characterized using flowprobe extractive analysis. Chemical images and profiles of dose response were effective in determining both the disposition and the time resolved intensities of drugs, i.e., highest in the 6 hr. post dose sample in all compartments of the analyzed lung lesions, though primarily accumulated in the caseum core; as well as other diagnostic compounds and metabolites of interest in various tissue and sub-tissue types. These data correlated well to other techniques pointing to the rapid and sensitive characteristics of the flowprobe analysis.
Monitoring the signal for calibrated concentration curves for drugs not bonded to tissue, it was noted that the integrated abundance for ion current could be linearly determined through tissue surfaces while lipid compounds associated with the tissue extracted from within the volume of the surface remained steady. Switching to experiments using dyes on and below soluble film laminates, analysis indicated clear correlations for volumes and surface areas at various depths of extraction, in a time resolved fashion, using the gentle flowprobe analysis. The variation of timescales for the permeation of various depths using the same solvent system for the disparate in situ extraction and ionization techniques can be used to draw conclusions about throughput and preferential utility for various applications.
Live-microorganism plate-based analysis has begun to generate a statistically significant number of species/strain profiles collected at various time points and stresses. 100 non-pathogenic colonies’ spectra sorted by species, yielded a few significant masses despite having the mixed assortment of spectral types with different solvents and media. A subset of these data can be applied toward strain-specific microbial classification. This specificity in profiling would be of particular interest for medical diagnostics. Summed E. coli data from both a wild type and a mutant strain has shown some distinctions by a data processing pipeline. Top down data dependent analysis, focusing on the secondary MS fragmentation clustering, will be discussed.
In all, these results point to various challenges and opportunities for direct extractive analysis workflow changes, to be discussed.
References & Acknowledgements:
1. Olson, Matthew T., Aparna Baxi, Mariam ElNaggar, Christopher Umbricht, Alfred L. Yergey, and William Clarke. "Morphologically compatible mass spectrometric analysis of lipids in cytological specimens." Journal of the American Society of Cytopathology 5, no. 1 (2016): 3-8.
2. Prideaux, Brendan, Mariam S. ElNaggar, Matthew Zimmerman, Justin M. Wiseman, Xiaohua Li, and Véronique Dartois. "Mass spectrometry imaging of levofloxacin distribution in TB-infected pulmonary lesions by MALDI-MSI and continuous liquid microjunction surface sampling." International Journal of Mass Spectrometry 377 (2015): 699-708.
3. ElNaggar, Mariam S., Brenden Prideaux, Veronique Dartois, and Justin M Wiseman. "Metabolic Imaging through Continuous In Situ Micro-extractions of Tissue Samples via Flowprobe Mass Spectrometry." Current Metabolomics 2, no. 2 (2014): 122-131.
4. Hsu, Cheng-Chih, Mariam S. ElNaggar, Yao Peng, Jinshu Fang, Laura M. Sanchez, Samantha J. Mascuch, Kirsten A. Møller et al. "Real-time metabolomics on living microorganisms using ambient electrospray ionization flow-probe." Analytical Chemistry 85, no. 15 (2013): 7014-7018.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Prosolia continues to employ me |
| Board Member | no | |
| Stock | yes | Prosolia continues to employ me |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |