25 research outputs found
Development of Advanced Optics and High Resolution Instrumentation for Mass Spectrometry Based Proteomics
Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain
spatial and chemical information of analytes on a wide variety of surfaces. The ability to
image biological analytes is an important tool in many areas of life science research,
including: the ability to map pharmaceutical drugs in targeted tissue, to spatially
determine the expression profile of specific proteins in healthy vs. diseased tissue states,
and to rapidly interrogate biomolecular microarrays. However, there are several avenues
for improving the imaging MS experiment for biological samples. Three significant
directions this work addresses include: (1) reducing chemical noise and increasing
analyte identification by developing sample preparation methodologies, (2) improving
the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a
spatially dynamic optical system, and (3) increasing both mass spectral resolution and
ion detection sensitivity by modifying a commercial time-of-flight (TOF) MS.
Firstly, sample methodology schemes presented in these studies consist of
obtaining both ?top-down? and ?bottom-up? information. In that, both intact mass and
peptide mass fingerprinting data can be obtained to increase protein identification. This sample methodology was optimized on protein microarrays in preparation for bio tissue
analysis. Other work consists of optimizing novel sample preparation strategies for
hydrated solid-supported lipid bilayer studies. Sample methods incorporating
nanomaterials for laser desorption/ionization illustrate the ability to perform selective
ionization of specific analytes. Specifically, our results suggest that silver nanoparticles
facilitate the selective ionization of olefin containing species (e.g., steroids, vitamins).
Secondly, an advanced optical design incorporating a spatially dynamic optical
scheme allows for laser beam expansion, homogenization, collimation, shaping, and
imaging. This spatially dynamic optical system allows user defined beam shapes,
decreases analysis times associated with mechanical movement of the sample stage, and
is capable of increasing the MS limits of detection by simultaneously irradiating multiple
spots.
Lastly, new data acquisition strategies (multiple anode detection schemes) were
incorporated into a commercial time-of-flight mass spectrometer to increase both
sensitivity and resolution in a matrix assisted laser desorption/ionization mass
spectrometer. The utility of this technique can be applied to many different samples,
where high mass spectral resolution allows for increased mass measurement accuracy
Metabolic consequences of interleukin-6 challenge in developing neurons and astroglia
Abstract Background: Maternal immune activation and subsequent interleukin-6 (IL-6) induction disrupt normal brain development and predispose the offspring to developing autism and schizophrenia. While several proteins have been identified as having some link to these developmental disorders, their prevalence is still small and their causative role, if any, is not well understood. However, understanding the metabolic consequences of environmental predisposing factors could shed light on disorders such as autism and schizophrenia. Methods: To gain a better understanding of the metabolic consequences of IL-6 exposure on developing central nervous system (CNS) cells, we separately exposed developing neuron and astroglia cultures to IL-6 for 2 hours while collecting effluent from our gravity-fed microfluidic chambers. By coupling microfluidic technologies to ultra-performance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS), we were able to characterize the metabolic response of these CNS cells to a narrow window of IL-6 exposure. Results: Our results revealed that 1) the use of this technology, due to its superb media volume:cell volume ratio, is ideally suited for analysis of cell-type-specific exometabolome signatures; 2) developing neurons have low secretory activity at baseline, while astroglia show strong metabolic activity; 3) both neurons and astroglia respond to IL-6 exposure in a cell type-specific fashion; 4) the astroglial response to IL-6 stimulation is predominantly characterized by increased levels of metabolites, while neurons mostly depress their metabolic activity; and 5) disturbances in glycerophospholipid metabolism and tryptophan/kynurenine metabolite secretion are two putative mechanisms by which IL-6 affects the developing nervous system. Conclusions: Our findings are potentially critical for understanding the mechanism by which IL-6 disrupts brain function, and they provide information about the molecular cascade that links maternal immune activation to developmental brain disorders
MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.
The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies
Coupling Supported Lipid Bilayer Electrophoresis with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging
Herein, we describe
a new analytical platform utilizing advances
in heterogeneous supported lipid bilayer (SLB) electrophoresis and
matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)
imaging. This platform allowed for the separation and visualization
of both charged and neutral lipid membrane components without the
need for extrinsic labels. A heterogeneous SLB was created using vesicles
containing monosialoganglioside GM1, disialoganglioside GD1b, POPC,
as well as the ortho and para isomers of Texas Red-DHPE. These components
were then separated electrophoretically into five resolved bands.
This represents the most complex separation by SLB electrophoresis
performed to date. The SLB samples were flash frozen in liquid ethane
and dried under vacuum before imaging with MALDI-MS. Fluorescence
microscopy was employed to confirm the position of the Texas Red labeled
lipids, which agreed well with the MALDI-MS imaging results. These
results clearly demonstrate this platform’s ability to isolate
and identify nonlabeled membrane components within an SLB
A Solution to Antifolate Resistance in Group B Streptococcus: Untargeted Metabolomics Identifies Human Milk Oligosaccharide-Induced Perturbations That Result in Potentiation of Trimethoprim
Group B Streptococcus is an important human pathogen that causes serious infections during pregnancy which can lead to chorioamnionitis, funisitis, premature rupture of gestational membranes, preterm birth, neonatal sepsis, and death. GBS is evolving antimicrobial resistance mechanisms, and the work presented in this paper provides evidence that prebiotics such as human milk oligosaccharides can act as adjuvants to restore the utility of antibiotics.Adjuvants can be used to potentiate the function of antibiotics whose efficacy has been reduced by acquired or intrinsic resistance. In the present study, we discovered that human milk oligosaccharides (HMOs) sensitize strains of group B Streptococcus (GBS) to trimethoprim (TMP), an antibiotic to which GBS is intrinsically resistant. Reductions in the MIC of TMP reached as high as 512-fold across a diverse panel of isolates. To better understand HMOs’ mechanism of action, we characterized the metabolic response of GBS to HMO treatment using ultrahigh-performance liquid chromatography–high-resolution tandem mass spectrometry (UPLC-HRMS/MS) analysis. These data showed that when challenged by HMOs, GBS undergoes significant perturbations in metabolic pathways related to the biosynthesis and incorporation of macromolecules involved in membrane construction. This study represents reports the metabolic characterization of a cell that is perturbed by HMOs
Genomic, transcriptomic, and metabolomic profiles of hiPSC-derived dopamine neurons from clinically discordant brothers with identical PRKN deletions
We previously reported on two brothers who carry identical compound heterozygous PRKN mutations yet present with significantly different Parkinson’s Disease (PD) clinical phenotypes. Juvenile cases demonstrate that PD is not necessarily an aging-associated disease. Indeed, evidence for a developmental component to PD pathogenesis is accumulating. Thus, we hypothesized that the presence of additional genetic modifiers, including genetic loci relevant to mesencephalic dopamine neuron development, could potentially contribute to the different clinical manifestations of the two brothers. We differentiated human-induced pluripotent stem cells (hiPSCs) derived from the two brothers into mesencephalic neural precursor cells and early postmitotic dopaminergic neurons and performed wholeexome sequencing and transcriptomic and metabolomic analyses. No significant differences in the expression of canonical dopamine neuron differentiation markers were observed. Yet our transcriptomic analysis revealed a significant downregulation of the expression of three neurodevelopmentally relevant cell adhesion molecules, CNTN6, CNTN4 and CHL1, in the cultures of the more severely affected brother. In addition, several HLA genes, known to play a role in neurodevelopment, were differentially regulated. The expression of EN2, a transcription factor crucial for mesencephalic dopamine neuron development, was also differentially regulated. We further identified differences in cellular processes relevant to dopamine metabolism. Lastly, wholeexome sequencing, transcriptomics and metabolomics data all revealed differences in glutathione (GSH) homeostasis, the dysregulation of which has been previously associated with PD. In summary, we identified genetic differences which could potentially, at least partially, contribute to the discordant clinical PD presentation of the two brothers