Quantitative Mass Spectrometry Imaging of Prostaglandins as Silver Ion Adducts with Nanospray Desorption Electrospray Ionization

Prostaglandins (PG) are an important class of lipid biomolecules that are essential in many biological processes, including inflammation and successful pregnancy. Despite a high bioactivity, physiological concentrations are typically low, which makes direct mass spectrometric analysis of endogenous PG species challenging. Consequently, there have not been any studies investigating PG localization to specific morphological regions in tissue sections using mass spectrometry imaging (MSI) techniques. Herein, we show that silver ions, added to the solvent used for nanospray desorption electrospray ionization (nano-DESI) MSI, enhances the ionization of PGs and enables nano-DESI MSI of several species in uterine tissue from day 4 pregnant mice. It was found that detection of [PG + Ag]⁺ ions increased the sensitivity by ∼30 times, when compared to [PG – H]⁻ ions. Further, the addition of isotopically labeled internal standards enabled generation of quantitative ion images for the detected PG species. Increased sensitivity and quantitative MSI enabled the first proof-of-principle results detailing PG localization in mouse uterus tissue sections. These results show that PG species primarily localized to cellular regions of the luminal epithelium and glandular epithelium in uterine tissue. Further, this study provides a unique scaffold for future studies investigating the PG distribution within biological tissue samples

XPO1, FASN and PDCD6IP abundance by immunohistochemistry on normal adjacent prostate (NAP), low-grade prostate cancer (Gleason score 3+3 = 6) and high grade prostate cancer (Gleason score 4+5 = 9).

<p>Representative pictures of the staining from 2 independent samples per group.</p

Comparison of proteins identified by Hosseini-Beheshti <i>et al.</i>, Sandvig <i>et al.</i> and this study visualized by a Venn diagram.

<p>The number of proteins identified in each study are a compilation of the cancer-derived exosomal proteins identified by MS-MS.</p

Validation of protein expression by Western blotting.

<p>All four exosome samples and their corresponding cell lines were used for validation. Furthermore, supernatant from the pelleted exosomes was used as a control. The selected proteins FASN, XPO1, CD9 and PDCD6IP, were tested with ENO1 and GAPDH as controls. PSA was tested to confirm it is secreted through alternative secretion pathway and therefore not present within exosomes. The nearest protein marker (kDa) is indicated for each blot.</p

Proteins expression differences.

<p>Proteins with significant abundance changes (>1.50 log2 fold) between prostate cancer and immortalized primary prostate epithelial cell lines.</p

Subcellular assignment of the proteins identified within the different samples in panel A.

<p>Exosomes from all four cell lines (PNT2C2, RWPE-1, PC346C, VCaP) contained 60% of cytoplasmic proteins and 25% of transmembrane proteins. B. The top seven functions of exosomal proteins according to Ingenuity Pathway Analysis. Fisher's exact test was applied to calculate significance (p-value<0.05).</p

Unsupervised hierarchical clustering of differentially abundant proteins (n = 263 proteins with >2 peptides) based on their MS-peak intensity values.

<p>Each exosome sample was analyzed in triplicate. Results were mean centered and log-transformed. Relative protein abundance is colored-coded with red corresponding to a relatively high abundance, green r corresponding to a relatively low abundance, and grey indicating missing abundance values.</p

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

Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of <i>Leucoagaricus gongylophorus</i>, the Fungal Symbiont of Leaf-Cutter Ants

<div><p>Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, <i>Leucoagaricus gongylophorus</i>, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by <i>L</i>. <i>gongylophorus</i> within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three <i>Atta cephalotes</i> colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the <i>L</i>. <i>gongylophorus</i> genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.</p></div