Discovering the N-Terminal Methylome by Repurposing of Proteomic Datasets.

Protein α-N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free α-amino group at protein N-termini. To systematically explore the extent of α-N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the α-N-terminal methylome. This repurposing approach found evidence of α-N-methylation of established and novel protein substrates with canonical N-terminal motifs of established α-N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, α-N-methylation of noncanonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of α-N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation and methylation, on amino acids at the first position. We tested three potentially methylated proteins and confirmed the α-N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating that proteomic searching for α-N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global α-N-terminal methylome investigations.

A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

High-fat-diet induced obesity increases the proportion of linoleic acyl residues in dam serum and milk and in suckling neonate circulation.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.

Multiple Reaction Monitoring Profiling (MRM-Profiling) of Lipids To Distinguish Strain-Level Differences in Microbial Resistance in Escherichia coli.

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of Escherichia coli cultured with amoxicillin or amoxicillin/clavulanate, a ß-lactam and ß-lactamase inhibitor, respectively. t tests, analysis of variance and receiver operating characteristic (ROC) curves were used to determine the significance of each MRM. Principal component analysis was applied to distinguish different strains cultured under conditions that allowed or disallowed development of bacterial resistance. The results demonstrate that MRM-Profiling distinguishes the lipid profiles of resistant and nonresistant E. coli strains.

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label-free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co-migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142.

The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N2 fixation (BNF), a process highly sensitive to oxygen. Previous work has focused on determining protein expression levels under different growth conditions. A major gap of our knowledge is an understanding on how these expressed proteins are assembled into complexes and organized into metabolic pathways, an area that has not been thoroughly investigated. Here, we combined size-exclusion chromatography (SEC) with label-free quantitative mass spectrometry (MS) and bioinformatics to characterize many protein complexes from Cyanothece 51142 cells grown under a 12 h light-dark cycle. We identified 1386 proteins in duplicate biological replicates, and 64% of those proteins were identified as putative complexes. Pairwise computational prediction of protein-protein interaction (PPI) identified 74 822 putative interactions, of which 2337 interactions were highly correlated with published protein coexpressions. Many sequential glycolytic and TCA cycle enzymes were identified as putative complexes. We also identified many membrane complexes that contain cytoplasmic domains. Subunits of NDH-1 complex eluted in a fraction with an approximate mass of ∼669 kDa, and subunits composition revealed coexistence of distinct forms of NDH-1 complex subunits responsible for respiration, electron flow, and CO2 uptake. The complex form of the phycocyanin beta subunit was nonphosphorylated, and the monomer form was phosphorylated at Ser20, suggesting phosphorylation-dependent deoligomerization of the phycocyanin beta subunit. This study provides an analytical platform for future studies to reveal how these complexes assemble and disassemble as a function of diurnal and circadian rhythms.

Diet Impacts Pre-implantation Histotroph Proteomes in Beef Cattle.

In ruminants, the period from fertilization to implantation is relatively prolonged, and the survival of embryos depends on uterine secretions known as histotroph. Our objective was to determine if the pre-breeding diet affected histotroph proteomes in beef cattle. Cows were assigned to one of four diets: a control diet (CON), a high-protein diet (PROT), a high-fat diet (OIL), or a high-protein and high-fat diet (PROT + OIL). After 185 days on these diets, an intravaginal progesterone implant (CIDR) was inserted for 7 days. At 9 days after CIDR removal, animals with a corpus luteum were selected ( n = 16; 4 per treatment). Proteins were isolated from the histotroph collected by uterine lavage and analyzed with liquid chromatography-tandem mass spectrometry. Over 2000 proteins were expressed ( n ≥ 3 cows per treatment), with 1239 proteins being common among all of the groups. There were 20, 37, 85, and 123 proteins unique to CON, PROT + OIL, PROT, and OIL, respectively. Relative to CON, 23, 14, and 51 proteins were differentially expressed in PROT + OIL, PROT, and OIL, respectively. Functional analysis found that 53% of histotroph proteins were categorized as extracellular exosome, 3.28% as cell-cell adhesion, and 17.4% in KEGG metabolic pathways. Differences in proteomes among treatments support the idea that pre-breeding diet affects histotroph. Understanding the impact of diet on histotroph proteins may help improve conception rates.

Multiple reaction monitoring (MRM)-profiling for biomarker discovery applied to human polycystic ovarian syndrome.

RATIONALE: We describe multiple reaction monitoring (MRM)-profiling, which provides accelerated discovery of discriminating molecular features, and its application to human polycystic ovary syndrome (PCOS) diagnosis. The discovery phase of the MRM-profiling seeks molecular features based on some prior knowledge of the chemical functional groups likely to be present in the sample. It does this through use of a limited number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of the discovery phase is a set of precursor/product transitions. In the screening phase these MRM transitions are used to interrogate multiple samples (hence the name MRM-profiling). METHODS: MRM-profiling was applied to follicular fluid samples of 22 controls and 29 clinically diagnosed PCOS patients. Representative samples were delivered by flow injection to a triple quadrupole mass spectrometer set to perform a number of pre-chosen and chemically specific neutral loss and/or precursor ion MS/MS scans. The output of this discovery phase was a set of 1012 precursor/product transitions. In the screening phase each individual sample was interrogated for these MRM transitions. Principal component analysis (PCA) and receiver operating characteristic (ROC) curves were used for statistical analysis. RESULTS: To evaluate the method's performance, half the samples were used to build a classification model (testing set) and half were blinded (validation set). Twenty transitions were used for the classification of the blind samples, most of them (N = 19) showed lower abundances in the PCOS group and corresponded to phosphatidylethanolamine (PE) and phosphatidylserine (PS) lipids. Agreement of 73% with clinical diagnosis was found when classifying the 26 blind samples. CONCLUSIONS: MRM-profiling is a supervised method characterized by its simplicity, speed and the absence of chromatographic separation. It can be used to rapidly isolate discriminating molecules in healthy/disease conditions by tailored screening of signals associated with hundreds of molecules in complex samples.

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-ß) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.

Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67.

Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal-host relationship is discussed.

Multi-omic data integration links deleted in breast cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response.

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

The gp82 surface molecule of Trypanosoma cruzi metacyclic forms.

Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the parasite forms from the insect vector that initiate infection in the mammalian host. Studies with metacyclic forms generated in vitro, as counterparts of insect-borne parasites, have shown that gp82 plays an essential role in host cell invasion and in the establishment of infection by the oral route. Among the gp82 properties relevant for infection are the gastric mucin-binding capacity and the ability to induce the target cell signaling cascades that result in actin cytoskeleton disruption and lysosome exocytosis, events that facilitate parasite internalization. The gp82 sequences from genetically divergent T. cruzi strains are highly conserved, displaying >90 % identity. Both the host cell-binding sites, as well as the gastric mucin-binding sequence of gp82, are localized in the C-terminal domain of the molecule. In the gp82 structure model, the main cell-binding site consists of an α-helix, which connects the N-terminal ß-propeller domain to the C-terminal ß-sandwich domain, where the second cell binding site is nested. The two cell binding sites are fully exposed on gp82 surface. Downstream and close to the α-helix is the gp82 gastric mucin-binding site, which is partially exposed. All available data support the notion that gp82 is structurally suited for metacyclic trypomastigote invasion of host cells and for initiating infection by the oral route.

KVFinder: steered identification of protein cavities as a PyMOL plugin.

BACKGROUND: The characterization of protein binding sites is a major challenge in computational biology. Proteins interact with a wide variety of molecules and understanding of such complex interactions is essential to gain deeper knowledge of protein function. Shape complementarity is known to be important in determining protein-ligand interactions. Furthermore, these protein structural features have been shown to be useful in assisting medicinal chemists during lead discovery and optimization. RESULTS: We developed KVFinder, a highly versatile and easy-to-use tool for cavity prospection and spatial characterization. KVFinder is a geometry-based method that has an innovative customization of the search space. This feature provides the possibility of cavity segmentation, which alongside with the large set of customizable parameters, allows detailed cavity analyses. Although the main focus of KVFinder is the steered prospection of cavities, we tested it against a benchmark dataset of 198 known drug targets in order to validate our software and compare it with some of the largely accepted methods. Using the one click mode, we performed better than most of the other methods, staying behind only of hybrid prospection methods. When using just one of KVFinder's customizable features, we were able to outperform all other compared methods. KVFinder is also user friendly, as it is available as a PyMOL plugin, or command-line version. CONCLUSION: KVFinder presents novel usability features, granting full customizable and highly detailed cavity prospection on proteins, alongside with a friendly graphical interface. KVFinder is freely available on http://lnbio.cnpem.br/bioinformatics/main/software/.

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans.

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

Integrated Proteomics Characterization of NLRP3 Inflammasome Inhibitor MCC950 in Monocytic Cell Line Confirms Direct MCC950 Engagement with Endogenous NLRP3.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.

Impact of Extraction Methods and Transportation Conditions on Lipid Profiles of Bovine Oocytes.

Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.

Insights into the organization of dorsal spinal cord pathways from an evolutionarily conserved raldh2 intronic enhancer.

Comparative studies of the tetrapod raldh2 (aldh1a2) gene, which encodes a retinoic acid (RA) synthesis enzyme, have led to the identification of a dorsal spinal cord enhancer. Enhancer activity is directed dorsally to the roof plate and dorsal-most (dI1) interneurons through predicted Tcf- and Cdx-homeodomain binding sites and is repressed ventrally via predicted Tgif homeobox and ventral Lim-homeodomain binding sites. Raldh2 and Math1/Cath1 expression in mouse and chicken highlights a novel, transient, endogenous Raldh2 expression domain in dI1 interneurons, which give rise to ascending circuits and intraspinal commissural interneurons, suggesting roles for RA in the ontogeny of spinocerebellar and intraspinal proprioceptive circuits. Consistent with expression of raldh2 in the dorsal interneurons of tetrapods, we also found that raldh2 is expressed in dorsal interneurons throughout the agnathan spinal cord, suggesting ancestral roles for RA signaling in the ontogenesis of intraspinal proprioception.

SUMOylation pathway in Trypanosoma cruzi: functional characterization and proteomic analysis of target proteins.

SUMOylation is a relevant protein post-translational modification in eukaryotes. The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO) is covalently linked to a lysine residue of the target protein by an isopeptide bond, through a mechanism that includes an E1-activating enzyme, an E2-conjugating enzyme, and transfer to the target, sometimes with the assistance of a ligase. The modification is reversed by a protease, also responsible for SUMO maturation. A number of proteins have been identified as SUMO targets, participating in the regulation of cell cycle progression, transcription, translation, ubiquitination, and DNA repair. In this study, we report that orthologous genes corresponding to the SUMOylation pathway are present in the etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the SUMOylation system is functionally active in this protozoan parasite, having the requirements for SUMO maturation and conjugation. Immunofluorescence analysis showed that T. cruzi SUMO (TcSUMO) is predominantly found in the nucleus. To identify SUMOylation targets and get an insight into their physiological roles we generated transfectant T. cruzi epimastigote lines expressing a double-tagged T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity chromatography. By two-dimensional liquid chromatography-mass spectrometry a total of 236 proteins with diverse biological functions were identified as potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically validated as a bona fide SUMOylation substrate. Proteomic studies in other organisms have reported that orthologs of putative T. cruzi SUMOylated proteins are similarly modified, indicating conserved functions for protein SUMOylation in this early divergent eukaryote.

Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry.

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.