Thermal Denaturation of Fresh Frozen Tissue Enhances Mass Spectrometry Detection of Peptides.

Thermal denaturation (TD), known as antigen retrieval, heats tissue samples in a buffered solution to expose protein epitopes. Thermal denaturation of formalin-fixed paraffin-embedded samples enhances on-tissue tryptic digestion, increasing peptide detection using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS). We investigated the tissue-dependent effects of TD on peptide MALDI IMS and liquid chromatography-tandem mass spectrometry signal in unfixed, frozen human colon, ovary, and pancreas tissue. In a triplicate experiment using time-of-flight, orbitrap, and Fourier-transform ion cyclotron resonance mass spectrometry platforms, we found that TD had a tissue-dependent effect on peptide signal, resulting in a (22.5%) improvement in peptide detection from the colon, a (73.3%) improvement in ovary tissue, and a (96.6%) improvement in pancreas tissue. Biochemical analysis of identified peptides shows that TD facilitates identification of hydrophobic peptides.

Clostridioides difficile ferrosome organelles combat nutritional immunity.

Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.

MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue.

Gangliosides are acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the analysis of isomeric lipid structures in situ. Here, we demonstrate the gas-phase separation and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a standard mixture of both isomers, a biological extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were determined in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.

Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

Uncovering Molecular Heterogeneity in the Kidney With Spatially Targeted Mass Spectrometry.

The kidney functions through the coordination of approximately one million multifunctional nephrons in 3-dimensional space. Molecular understanding of the kidney has relied on transcriptomic, proteomic, and metabolomic analyses of kidney homogenate, but these approaches do not resolve cellular identity and spatial context. Mass spectrometry analysis of isolated cells retains cellular identity but not information regarding its cellular neighborhood and extracellular matrix. Spatially targeted mass spectrometry is uniquely suited to molecularly characterize kidney tissue while retaining in situ cellular context. This review summarizes advances in methodology and technology for spatially targeted mass spectrometry analysis of kidney tissue. Profiling technologies such as laser capture microdissection (LCM) coupled to liquid chromatography tandem mass spectrometry provide deep molecular coverage of specific tissue regions, while imaging technologies such as matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) molecularly profile regularly spaced tissue regions with greater spatial resolution. These technologies individually have furthered our understanding of heterogeneity in nephron regions such as glomeruli and proximal tubules, and their combination is expected to profoundly expand our knowledge of the kidney in health and disease.

RNA aptamer capture of macromolecular complexes for mass spectrometry analysis.

Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA-MS (aptamer affinity-mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA-MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.

Peptidomics Approaches for the Identification of Bioactive Molecules from Diaphorina citri.

Huanglongbing (HLB), a deadly citrus disease, is primarily associated with Candidatus Liberibacter asiaticus (CLas) and spread by the hemipteran insect Diaphorina citri. Control strategies to combat HLB are urgently needed. In this work, we developed and compared workflows for the extraction of the D. citri peptidome, a dynamic set of polypeptides produced by proteolysis and other cellular processes. High-resolution mass spectrometry revealed bias among methods reflecting the physiochemical properties of the peptides: while TCA/acetone-based methods resulted in enrichment of C-terminally amidated peptides, a modification characteristic of bioactive peptides, larger peptides were overrepresented in the aqueous phase of chloroform/methanol extracts, possibly indicative of reduced co-analytical degradation during sample preparation. Parallel reaction monitoring (PRM) was used to validate the structure and upregulation of peptides derived from hemocyanin, a D. citri immune system protein, in insects reared on healthy and CLas-infected trees. Mining of the data sets also revealed 122 candidate neuropeptides, including PK/PBAN family neuropeptides and kinins, biostable analogs of which have known insecticidal properties. Taken together, this information yields new, in-depth insights into peptidomics methodology. Additionally, the putative neuropeptides identified may lead to psyllid mortality if applied to or expressed in citrus, consequently blocking the spread of HLB disease in citrus groves.

Lessons from One Fastidious Bacterium to Another: What Can We Learn about Liberibacter Species from Xylella fastidiosa.

Huanglongbing is causing economic devastation to the citrus industry in Florida, and threatens the industry everywhere the bacterial pathogens in the Candidatus Liberibacter genus and their insect vectors are found. Bacteria in the genus cannot be cultured and no durable strategy is available for growers to control plant infection or pathogen transmission. However, scientists and grape growers were once in a comparable situation after the emergence of Pierce's disease, which is caused by Xylella fastidiosa and spread by its hemipteran insect vector. Proactive quarantine and vector control measures coupled with interdisciplinary data-driven science established control of this devastating disease and pushed the frontiers of knowledge in the plant pathology and vector biology fields. Our review highlights the successful strategies used to understand and control X. fastidiosa and their potential applicability to the liberibacters associated with citrus greening, with a focus on the interactions between bacterial pathogen and insect vector. By placing the study of Candidatus Liberibacter spp. within the current and historical context of another fastidious emergent plant pathogen, future basic and applied research to develop control strategies can be prioritized.

Candidatus Liberibacter asiaticus Minimally Alters Expression of Immunity and Metabolism Proteins in Hemolymph of Diaphorina citri, the Insect Vector of Huanglongbing.

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ' Candidatus Liberibacter asiaticus' ( CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.

Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut.

Huanglongbing, or citrus greening disease, is an economically devastating bacterial disease of citrus. It is associated with infection by the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP). For insect transmission to occur, CLas must be ingested during feeding on infected phloem sap and cross the gut barrier to gain entry into the insect vector. To investigate the effects of CLas exposure at the gut-pathogen interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and proteome, respectively, of ACP gut tissue. CLas exposure resulted in changes in pathways involving the TCA cycle, iron metabolism, insecticide resistance and the insect's immune system. We identified 83 long non-coding RNAs that are responsive to CLas, two of which appear to be specific to the ACP. Proteomics analysis also enabled us to determine that Wolbachia, a symbiont of the ACP, undergoes proteome regulation when CLas is present. Fluorescent in situ hybridization (FISH) confirmed that Wolbachia and CLas inhabit the same ACP gut cells, but do not co-localize within those cells. Wolbachia cells are prevalent throughout the gut epithelial cell cytoplasm, and Wolbachia titer is more variable in the guts of CLas exposed insects. CLas is detected on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei. Our study provides a snapshot of how the psyllid gut copes with CLas exposure and provides information on pathways and proteins for targeted disruption of CLas-vector interactions at the gut interface.

The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance.

BACKGROUND: The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is among the 100 worst invasive species in the world. As one of the most important crop pests and virus vectors, B. tabaci causes substantial crop losses and poses a serious threat to global food security. RESULTS: We report the 615-Mb high-quality genome sequence of B. tabaci Middle East-Asia Minor 1 (MEAM1), the first genome sequence in the Aleyrodidae family, which contains 15,664 protein-coding genes. The B. tabaci genome is highly divergent from other sequenced hemipteran genomes, sharing no detectable synteny. A number of known detoxification gene families, including cytochrome P450s and UDP-glucuronosyltransferases, are significantly expanded in B. tabaci. Other expanded gene families, including cathepsins, large clusters of tandemly duplicated B. tabaci-specific genes, and phosphatidylethanolamine-binding proteins (PEBPs), were found to be associated with virus acquisition and transmission and/or insecticide resistance, likely contributing to the global invasiveness and efficient virus transmission capacity of B. tabaci. The presence of 142 horizontally transferred genes from bacteria or fungi in the B. tabaci genome, including genes encoding hopanoid/sterol synthesis and xenobiotic detoxification enzymes that are not present in other insects, offers novel insights into the unique biological adaptations of this insect such as polyphagy and insecticide resistance. Interestingly, two adjacent bacterial pantothenate biosynthesis genes, panB and panC, have been co-transferred into B. tabaci and fused into a single gene that has acquired introns during its evolution. CONCLUSIONS: The B. tabaci genome contains numerous genetic novelties, including expansions in gene families associated with insecticide resistance, detoxification and virus transmission, as well as numerous horizontally transferred genes from bacteria and fungi. We believe these novelties likely have shaped B. tabaci as a highly invasive polyphagous crop pest and efficient vector of plant viruses. The genome serves as a reference for resolving the B. tabaci cryptic species complex, understanding fundamental biological novelties, and providing valuable genetic information to assist the development of novel strategies for controlling whiteflies and the viruses they transmit.

Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles' Heel of the Citrus Greening Insect Vector.

'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including 'Candidatus Profftella armatura', are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.