7 research outputs found
Applying Multimodal Mass Spectrometry to Image Tumors Undergoing Ferroptosis Following <i>In Vivo</i> Treatment with a Ferroptosis Inducer
Epithelial ovarian
cancer (EOC) is the most common form of ovarian
cancer. The poor prognosis generally associated with this disease
has led to the search for improved therapies such as ferroptosis-inducing
agents. Ferroptosis is a form of regulated cell death that is dependent
on iron and is characterized by lipid peroxidation. Precise mapping
of lipids and iron within tumors exposed to ferroptosis-inducing agents
may provide insight into processes of ferroptosis in vivo and ultimately assist in the optimal deployment of ferroptosis inducers
in cancer therapy. In this work, we present a method for combining
matrix-assisted laser desorption/ionization (MALDI) mass spectrometry
imaging (MSI) with secondary ion mass spectrometry (SIMS) to analyze
changes in spatial lipidomics and metal composition, respectively,
in ovarian tumors following exposure to a ferroptosis inducer. Tumors
were obtained by injecting human ovarian cancer tumor-initiating cells
into mice, followed by treatment with the ferroptosis inducer erastin.
SIMS imaging detected iron accumulation in the tumor tissue, and sequential
MALDI-MS imaging of the same tissue section displayed two chemically
distinct regions of lipids. One region was associated with the iron-rich
area detected with SIMS, and the other region encompassed the remainder
of the tissue section. Bulk lipidomics confirmed the lipid assignments
putatively assigned from the MALDI-MS data. Overall, we demonstrate
the ability of multimodal MSI to identify the spatial locations of
iron and lipids in the same tissue section and associate these regions
with clinical pathology
Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer’s Disease Across Distinct Brain Regions
Alzheimer’s disease (AD) is a neurodegenerative
disease
with a complex etiology influenced by confounding factors such as
genetic polymorphisms, age, sex, and race. Traditionally, AD research
has not prioritized these influences, resulting in dramatically skewed
cohorts such as three times the number of Apolipoprotein E (APOE)
ε4-allele carriers in AD relative to healthy cohorts. Thus,
the resulting molecular changes in AD have previously been complicated
by the influence of apolipoprotein E disparities. To explore how apolipoprotein
E polymorphism influences AD progression, 62 post-mortem patients
consisting of 33 AD and 29 controls (Ctrl) were studied to balance
the number of ε4-allele carriers and facilitate a molecular
comparison of the apolipoprotein E genotype. Lipid and protein perturbations
were assessed across AD diagnosed brains compared to Ctrl brains,
ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s
and APOE4– for non-ε4 carriers), and differences in ε3ε3
and ε3ε4 Ctrl brains across two brain regions (frontal
cortex (FCX) and cerebellum (CBM)). The region-specific influences
of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction
and cell proteostasis at the core of AD pathophysiology in the post-mortem
brains, indicating these two processes may be influenced by genotypic
differences and brain morphology
Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer’s Disease Across Distinct Brain Regions
Alzheimer’s disease (AD) is a neurodegenerative
disease
with a complex etiology influenced by confounding factors such as
genetic polymorphisms, age, sex, and race. Traditionally, AD research
has not prioritized these influences, resulting in dramatically skewed
cohorts such as three times the number of Apolipoprotein E (APOE)
ε4-allele carriers in AD relative to healthy cohorts. Thus,
the resulting molecular changes in AD have previously been complicated
by the influence of apolipoprotein E disparities. To explore how apolipoprotein
E polymorphism influences AD progression, 62 post-mortem patients
consisting of 33 AD and 29 controls (Ctrl) were studied to balance
the number of ε4-allele carriers and facilitate a molecular
comparison of the apolipoprotein E genotype. Lipid and protein perturbations
were assessed across AD diagnosed brains compared to Ctrl brains,
ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s
and APOE4– for non-ε4 carriers), and differences in ε3ε3
and ε3ε4 Ctrl brains across two brain regions (frontal
cortex (FCX) and cerebellum (CBM)). The region-specific influences
of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction
and cell proteostasis at the core of AD pathophysiology in the post-mortem
brains, indicating these two processes may be influenced by genotypic
differences and brain morphology
Ion Mobility Spectrometry for Enhanced Omic Analyses (22nd Annual Lorne Proteomics Symposium 2017)
We would like to increase throughput of measurements and IMS-MS analyses are able to detect high feature numbers with fast LC separations or no LC separations at all. IMS-TOF MS provides greater dynamic range of detection relative to trapping (e.g. Orbitrap) instruments. IMS adds complementary information to MS measurements which helps lower false discovery rates and separates isomers. Detection of structural changes in peptides/ proteins that can help characterize specific disease states (structural biomarkers)<div><br></div
Automated Solid Phase Extractions Coupled with Ion Mobility-Mass Spectrometry for Rapid Metabolic Screening.(ASMS 2016)
<p>Metabolomic analyses of complex
plasma and urine samples present numerous analytical challenges, such as
isomeric indistinguishability and inadequate measurement throughput. Ion
mobility separations (IMS) minimize these limitations by providing high
throughput structurally informative analyses, and when combined with mass
spectrometry (MS) measurements, the multidimensional IMS-MS analyses provide in
depth metabolite characterization. However, ionization suppression is typically
observed in ESI-IMS-MS direct injection studies of plasma and urine due to the
numerous components and their concentrations. Rapid separations and sample
cleanup prior to IMS-MS analyses can avoid suppression and enable broader molecular
coverage. In this study, we explored the use of automated solid phase
extractions (SPE) coupled with IMS-MS to rapidly analyze plasma and urine samples.
</p
Diacyltransferase Activity and Chain Length Specificity of <i>Mycobacterium tuberculosis</i> PapA5 in the Synthesis of Alkyl β‑Diol Lipids
Although they are classified as Gram-positive
bacteria, Corynebacterineae
possess an asymmetric outer membrane that imparts structural and thereby
physiological similarity to more distantly related Gram-negative bacteria.
Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer
membrane of Corynebacterineae have been associated with the virulence
of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl
esters known as dimycocerosates (DIMs) are significantly attenuated
in model infections. The resultant interest in the biosynthetic pathway
of these unusual virulence factors has led to the elucidation of many
of the steps leading to the final esterification of the alkyl β-diol,
phthiocerol, with branched-chain fatty acids known as mycocerosates.
PapA5 is an acyltransferase implicated in these final reactions. Here,
we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis
by demonstrating its dual esterification activity and chain-length
preference using synthetic alkyl β-diol substrate analogues.
By applying these analogues to a series of PapA5 mutants, we also
revise a model for the substrate binding within PapA5. Finally, we
demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5
on three overlapping Thr residues and that a fourth Thr is unique
to PknE phosphorylation. These results clarify the DIM biosynthetic
pathway and indicate post-translational modifications that warrant
further elucidation for their roles in the regulation of DIM biosynthesis
Table1_An optimized approach and inflation media for obtaining complimentary mass spectrometry-based omics data from human lung tissue.DOCX
Human disease states are biomolecularly multifaceted and can span across phenotypic states, therefore it is important to understand diseases on all levels, across cell types, and within and across microanatomical tissue compartments. To obtain an accurate and representative view of the molecular landscape within human lungs, this fragile tissue must be inflated and embedded to maintain spatial fidelity of the location of molecules and minimize molecular degradation for molecular imaging experiments. Here, we evaluated agarose inflation and carboxymethyl cellulose embedding media and determined effective tissue preparation protocols for performing bulk and spatial mass spectrometry-based omics measurements. Mass spectrometry imaging methods were optimized to boost the number of annotatable molecules in agarose inflated lung samples. This optimized protocol permitted the observation of unique lipid distributions within several airway regions in the lung tissue block. Laser capture microdissection of these airway regions followed by high-resolution proteomic analysis allowed us to begin linking the lipidome with the proteome in a spatially resolved manner, where we observed proteins with high abundance specifically localized to the airway regions. We also compared our mass spectrometry results to lung tissue samples preserved using two other inflation/embedding media, but we identified several pitfalls with the sample preparation steps using this preservation method. Overall, we demonstrated the versatility of the inflation method, and we can start to reveal how the metabolome, lipidome, and proteome are connected spatially in human lungs and across disease states through a variety of different experiments.</p