PubMed

Microbiol Spectr. 2023 Feb 14:e0468822. doi: 10.1128/spectrum.04688-22. Online ahead of print.ABSTRACTBabesia divergens is an apicomplexan parasite that infects human red blood cells (RBCs), initiating cycles of invasion, replication, and egress, resulting in extensive metabolic modification of the host cells. Babesia is an auxotroph for most of the nutrients required to sustain these cycles. There are currently limited studies on the biochemical pathways that support these critical processes, necessitating the high-resolution global metabolomics approach described here to uncover the metabolic interactions between parasite and host RBC. Our results reveal an extensive parasite-mediated modulation of RBC metabolite levels of all classes, including lipids, amino acids, carbohydrates, and nucleotides, with numerous metabolic species varying in proportion to the level of infection. Many of these molecules are scavenged from the host RBCs. This is in accord with the needs of a rapidly proliferating parasite with limited biosynthetic capabilities. Probing these pathways in depth, we used growth inhibition assays to quantitate parasite susceptibility to drugs targeting these pathways and stimulated emission depletion (STED) microscopy to obtain high-resolution images of drug-treated parasites to correlate changes in morphology with specific metabolic blocks in order to validate the data generated by the untargeted metabolomics platform. Thus, interruption of cholesterol scavenging from the host cell led to premature parasite egress, while chemical targeting of the hydrolysis of acyl glycerides led to the buildup of malformed parasites that could not successfully egress. This is the first report detailing the global metabolomic profile of the B. divergens-infected RBC. Besides deciphering diverse aspects of the host-parasite relationship, our results can be exploited by others to uncover further drug targets in the host-parasite biochemical network. IMPORTANCE Human babesiosis is caused by apicomplexan parasites of the Babesia genus and is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations. Through its continuous cycles of invasion, proliferation, and egress, B. divergens radically changes the metabolic environment of the host red blood cell, allowing us opportunities to study potential chemical vulnerabilities that can be targeted by drugs. This is the first global metabolomic profiling of Babesia-infected human red blood cells, and our analysis revealed perturbation in all biomolecular classes at levels proportional to the level of infection. In particular, lipids and energy flux pathways in the host cell were altered by infection. We validated the changes in key metabolic pathways by performing inhibition assays accompanied by high-resolution microscopy. Overall, this global metabolomics analysis of Babesia-infected red blood cells has helped to uncover novel aspects of parasite biology and identified potential biochemical pathways that can be targeted for chemotherapeutic intervention.PMID:36786651 | DOI:10.1128/spectrum.04688-22

Microbiol Spectr. 2023 Feb 14:e0135022. doi: 10.1128/spectrum.01350-22. Online ahead of print.ABSTRACTThe seed-borne microbiota and seed metabolites of the grass Achnatherum inebrians, either host to Epichloë gansuensis (endophyte infected [EI]) or endophyte free (EF), were investigated. This study determined the microbial communities both within the seed (endophytic) and on the seed surface (epiphytic) and of the protective glumes by using Illumina sequencing technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. In addition, metabolites of seeds and glumes were detected using liquid chromatography-mass spectrometry (LC-MS). Unlike with the seeds of EF plants, the presence of E. gansuensis resulted in significant changes in the content of 108 seed and 31 glume metabolites. A total of 319 significant correlations occurred between seed-borne microbiota and seed metabolites; these correlations comprised 163 (147 bacterial and 16 fungal) positive correlations and 156 (136 bacterial and 20 fungal) negative correlations. Meanwhile, there were 42 significant correlations between glume microbiota and metabolites; these correlations comprised 28 positive (10 bacterial and 18 fungal) and 14 negative (9 bacterial and 5 fungal) correlations. The presence of E. gansuensis endophyte altered the communities and diversities of seed-borne microbes and altered the composition and content of seed metabolites, and there were many close and complex relationships between microbes and metabolites. IMPORTANCE The present study was to investigate seed-borne microbiota and seed metabolites in Achnatherum inebrians using high-throughput sequencing and LC-MS technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. Compared with endophyte-free plants, the content of 108 seed and 31 glume metabolites of endophyte-infected plants was significantly changed. There were 319 significant correlations between seed-borne microbiota and seed metabolites and 42 significant correlations between glume microbiota and metabolites.PMID:36786621 | DOI:10.1128/spectrum.01350-22

mBio. 2023 Feb 14:e0342422. doi: 10.1128/mbio.03424-22. Online ahead of print.ABSTRACTMicrobes possess conserved microbe-associated molecular patterns (MAMPs) that are recognized by plant receptors to induce pattern-triggered immunity (PTI). Despite containing the same MAMPs as pathogens, commensals thrive in the plant rhizosphere microbiome, indicating they must suppress or evade host immunity. Previous work found that bacterial-secreted gluconic acid is sufficient to suppress PTI. Here, we show that gluconic acid biosynthesis is not necessary for immunity suppression by the beneficial bacterial strain Pseudomonas simiae WCS417. We performed a forward genetic screen with EMS-mutagenized P. simiae WCS417 and a flagellin-inducible CYP71A12pro:GUS reporter as a PTI readout. We identified a loss of function mutant in ornithine carbamoyltransferase argF, which is required for ornithine conversion to arginine, that cannot suppress PTI or acidify the rhizosphere. Fungal pathogens use alkalization through production of ammonia and glutamate, and arginine biosynthetic precursors, to promote their own growth and virulence. While a ΔargF mutant has a growth defect in the rhizosphere, we found that restoring growth with exogenous arginine resulted in rhizosphere alkalization in a mutant that cannot make gluconic acid, indicating that arginine biosynthesis is required for both growth and acidification. Furthermore, blocking bacterial arginine, glutamine, or proline biosynthesis through genetic mutations or feedback inhibition by adding corresponding amino acids, resulted in rhizosphere alkalization. Untargeted metabolomics determined that ornithine, an alkaline molecule, accumulates under conditions associated with rhizosphere alkalization. Our findings show that bacterial amino acid biosynthesis contributes to acidification by preventing accumulation of ornithine and the resulting alkalization. IMPORTANCE Understanding how microbiota evade and suppress host immunity is critical to our knowledge of how beneficial microbes persist in association with a host. Prior work has shown that secretion of organic acids by beneficial microbes is sufficient to suppress plant immunity. This work shows that microbial amino acid metabolism is not only critical for growth in the plant rhizosphere microbiome, but also for regulation of plant rhizosphere pH, and, consequentially, regulation of plant immunity. We found that, in the absence of microbial glutamate and arginine metabolism, rhizosphere alkalization and microbial overgrowth occurs. Collectively, our findings suggest that, by regulating nutrient availability, plants have the potential to regulate their immune homeostasis in the rhizosphere microbiome.PMID:36786577 | DOI:10.1128/mbio.03424-22

Microbiol Spectr. 2023 Feb 14:e0395722. doi: 10.1128/spectrum.03957-22. Online ahead of print.ABSTRACTNematode-trapping (NT) fungi are a unique group of carnivorous microorganisms that can capture and digest nematodes by producing ingenious trapping devices (traps). Arthrobotrys oligospora, a representative NT fungus, can develop adhesive three-dimensional networks for nematode predation. Hyphal fusion is indispensable for the trap formation of A. oligospora. Here, we characterized an orthologous Ste12 protein (AoSte12) in A. oligospora via gene disruption, DNA affinity purification sequencing (DAP-Seq), and multi-omics approaches. The disruption of the Aoste12 gene caused an increase in hyphal fusion and resulted in defects in mycelial growth, conidiation, trap morphology, and stress resistance, as well as reducing the number of nuclei and lipid droplet accumulation. Moreover, transcriptome and DAP-Seq analysis revealed that AoSte12 was involved in cellular processes associated with growth, cell fusion, the tricarboxylic acid cycle, vesicles, actin filaments, and lipid metabolism. In addition, combining metabolome with transcriptome and DAP-Seq analysis indicated that AoSte12 was involved in the mitogen-activated protein kinase signaling pathway, lipid metabolism, and secondary metabolites. A yeast two-hybrid assay revealed that AoSte12 can interact with diverse proteins, such as the MAK-2 orthologue protein Fus3, the vacuolar sorting protein Pep3, and UDP-glycosyltransferase. Our results suggest that AoSte12 plays an indispensable role in hyphal fusion and thus regulates sporulation and trap morphogenesis. These results provide deep insights into the connection between hyphal fusion and trap formation in NT fungi. IMPORTANCE Nematode-trapping (NT) fungi are an important natural enemy of nematodes and can capture their prey by producing traps. Hyphal anastomosis and fusion are important for mycelial growth and the colony morphological development of filamentous fungi and are also crucial for the trap morphogenesis of NT fungi. Arthrobotrys oligospora can form complex three-dimensional networks (traps) when sensing the presence of nematodes. This study revealed that AoSte12 is indispensable for hyphal fusion and that it regulates mycelial growth, conidiation, trap morphogenesis, stress resistance, the number of nuclei, and lipid droplet accumulation in A. oligospora. In addition, DNA affinity purification sequencing, transcriptome, and metabolome analyses further revealed that AoSte12 is involved in the mitogen-activated protein kinase pathway, lipid metabolism, and secondary metabolism. Overall, these findings expand the important role of AoSte12 in NT fungus A. oligospora and provide a broad foundation for elucidating the regulatory mechanism of trap development and the lifestyle transitions of pathogenic fungi.PMID:36786575 | DOI:10.1128/spectrum.03957-22

Food Funct. 2023 Feb 14. doi: 10.1039/d2fo03885d. Online ahead of print.ABSTRACTIncreasing studies have demonstrated that ginsenoside Rg3 (Rg3) plays an important role in the prevention and treatment of various diseases, including allergic lower airway inflammation such as asthma. To investigate the role of Rg3 in allergic upper airway disease, the effect and therapeutic mechanism of Rg3 in allergic rhinitis (AR) were studied. Ovalbumin-induced AR model mice were intragastrically administered with Rg3. Nasal symptoms, levels of IgE, IL-4, IL-5, IL-13, SOD and MDA in serum, and histopathological analysis of nasal mucosa were used to evaluate the effect of Rg3 on ameliorating AR in mice. Moreover, nasal mucosa samples from the normal control group, AR model group and high dosage of Rg3 were collected to perform omics analysis. The differentially expressed genes and significantly changed metabolites were screened based on transcriptomics and metabolomics analyses, respectively. Integrative analysis was further performed to confirm the hub genes, metabolites and pathways. After Rg3 intervention, the nasal symptoms and inflammatory infiltration were effectively improved, the levels of IgE, IL-4, IL-5, IL-13 and MDA were significantly reduced, and the level of SOD was obviously increased. The results of the qRT-PCR assay complemented the transcriptomic findings. Integrated analysis showed that Rg3 played an anti-AR role mainly by regulating the interaction network, which was constructed by 12 genes, 8 metabolites and 4 pathways. Our findings suggested that Rg3 had a therapeutic effect on ovalbumin-induced AR in mice by inhibiting inflammation development and reducing oxidative stress. The present study could provide a potential natural agent for the treatment of AR.PMID:36786409 | DOI:10.1039/d2fo03885d

Bioinformatics. 2023 Feb 14:btad088. doi: 10.1093/bioinformatics/btad088. Online ahead of print.ABSTRACTMOTIVATION: Plasma ionization is rapidly gaining popularity for MS based studies of volatiles and aerosols. However, data from plasma ionization is delicate to interpret as competing ionization pathways in the plasma create numerous ion species. There is no tool for detection of adducts and in-source fragments from plasma ionization data yet, which makes data evaluation ambiguous.SUMMARY: We developed DBDIpy, a Python library for processing and formal analysis of untargeted, time-sensitive plasma ionization MS datasets. Its core functionality lies in the identification of in-source fragments and identification of rivaling ionization pathways of the same analytes in time-sensitive datasets. It further contains elementary functions for processing of untargeted metabolomics data and interfaces to an established ecosystem for analysis of MS data in Python.AVAILABILITY AND IMPLEMENTATION: DBDIpy is implemented in Python (Version ≥ 3.7) and can be downloaded from PyPI the Python package repository (https://pypi.org/project/DBDIpy) or from GitHub (https://github.com/leopold-weidner/DBDIpy).PMID:36786403 | DOI:10.1093/bioinformatics/btad088

Allergy. 2023 Feb 13. doi: 10.1111/all.15672. Online ahead of print.NO ABSTRACTPMID:36785923 | DOI:10.1111/all.15672

Oxid Med Cell Longev. 2023 Feb 4;2023:4416410. doi: 10.1155/2023/4416410. eCollection 2023.ABSTRACTAging is a complex process characterized by progressive loss of functional abilities due to the accumulation of molecular damages. Metabolomics could offer novel insights into the predictors and mechanisms of aging. This cross-sectional study is aimed at identifying age-associated plasma metabolome in a Malay population. A total of 146 (90 females) healthy participants aged 28-69 were selected for the study. Untargeted metabolomics profiling was performed using liquid chromatography-tandem mass spectrometry. Association analysis was based on the general linear model. Gender-associated metabolites were adjusted for age, while age-associated metabolites were adjusted for gender or analyzed in a gender-stratified manner. Gender-associated metabolites such as 4-hydroxyphenyllactic acid, carnitine, cortisol, and testosterone sulfate showed higher levels in males than females. Deoxycholic acid and hippuric acid were among the metabolites with a positive association with age after being adjusted for gender, while 9(E),11(E)-conjugated linoleic acid, cortisol, and nicotinamide were negatively associated with age. In gender-stratified analysis, glutamine was one of the common metabolites that showed a direct association with age in both genders, while metabolites such as 11-deoxy prostaglandin F2β, guanosine monophosphate, and testosterone sulfate were inversely associated with age in males and females. This study reveals several age-associated metabolites in Malays that could reflect the changes in metabolisms during aging and may be used to discern the risk of geriatric syndromes and disorders later. Further studies are required to determine the interplay between these metabolites and environmental factors on the functional outcomes during aging.PMID:36785791 | PMC:PMC9922189 | DOI:10.1155/2023/4416410

PeerJ. 2023 Feb 8;11:e14869. doi: 10.7717/peerj.14869. eCollection 2023.ABSTRACTSugar metabolites not only act as the key compounds in tea plant response to stress but are also critical for tea quality formation during the post-harvest processing of tea leaves. However, the mechanisms by which sugar metabolites in post-harvest tea leaves respond to mechanical stress are unclear. In this study, we aimed to investigate the effects of mechanical stress on saccharide metabolites and related post-harvest tea genes. Withered (C15) and mechanically-stressed (V15) for 15 min Oolong tea leaves were used for metabolome and transcriptome sequencing analyses. We identified a total of 19 sugar metabolites, most of which increased in C15 and V15. A total of 69 genes related to sugar metabolism were identified using transcriptome analysis, most of which were down-regulated in C15 and V15. To further understand the relationship between the down-regulated genes and sugar metabolites, we analyzed the sucrose and starch, galactose, and glycolysis metabolic pathways, and found that several key genes of invertase (INV), α-amylase (AMY), β-amylase (BMY), aldose 1-epimerase (AEP), and α-galactosidase (AGAL) were down-regulated. This inhibited the hydrolysis of sugars and might have contributed to the enrichment of galactose and D-mannose in V15. Additionally, galactinol synthase (Gols), raffinose synthase (RS), hexokinase (HXK), 6-phosphofructokinase 1 (PFK-1), and pyruvate kinase (PK) genes were significantly upregulated in V15, promoting the accumulation of D-fructose-6-phosphate (D-Fru-6P), D-glucose-6-phosphate (D-glu-6P), and D-glucose. Transcriptome and metabolome association analysis showed that the glycolysis pathway was enhanced and the hydrolysis rate of sugars related to hemicellulose synthesis slowed in response to mechanical stress. In this study, we explored the role of sugar in the response of post-harvest tea leaves to mechanical stress by analyzing differences in the expression of sugar metabolites and related genes. Our results improve the understanding of post-harvest tea's resistance to mechanical stress and the associated mechanism of sugar metabolism. The resulting treatment may be used to control the quality of Oolong tea.PMID:36785711 | PMC:PMC9921968 | DOI:10.7717/peerj.14869

Biophys J. 2023 Feb 10;122(3S1):93a-94a. doi: 10.1016/j.bpj.2022.11.702.NO ABSTRACTPMID:36785094 | DOI:10.1016/j.bpj.2022.11.702

BMC Plant Biol. 2023 Feb 13;23(1):89. doi: 10.1186/s12870-023-04093-2.ABSTRACTBACKGROUND: Leaves of tobacco (Nicotiana tabacum L.) are flue-cured to use as a key industrial supply in various parts of the world. The quality of tobacco leaves is dependent on chemical components and their proportions. Generally, the stem attached to tobacco leaf is detached before curing. However, the leaf stem remains green for an extended period of time (as compared to leaf) during flue-curing. Hence, it is expected to affect the quality of tobacco's final product.RESULTS: To understand the impact of the green stem of leaf on the metabolome of flue-cured tobacco, we employed a broad targeted metabolomics approach. We selected two tobacco cultivars (Yun87 and K326) and cultivated them in five geographic locations in China. For flue-curing, leaves were harvested without a stem (L) or with an attached stem (SPL). After metabolome analysis, a total of 1027 metabolites were annotated in these samples. A variable number of metabolites were differentially accumulated between both types of leaves (depending on geographic location or cultivar) representing an influence of environment or genotype. Interestingly, only 68 metabolites were differentially accumulated between L and SPL samples irrespective of the cultivar or geographic location. These differentially accumulated metabolites belonged to major groups of primary and secondary metabolites. We have discussed the importance of identified metabolites in terms of carbon, nitrogen, and polyphenolic metabolism.CONCLUSION: The present research is the first comprehensive description of several metabolites in tobacco leaves related to the contribution of leaf stem. The current study opens novel prospects for investigating the potential of such metabolites in improving the quality of flue-cured tobacco.PMID:36782114 | DOI:10.1186/s12870-023-04093-2

Diabet Med. 2023 Feb 13:e15064. doi: 10.1111/dme.15064. Online ahead of print.ABSTRACTOBJECTIVE: The objective of this scoping review is to evaluate the current biomarkers used in the assessment of adverse cardiac remodelling in people with diabetes mellitus (DM) and in the prognosis of subsequent cardiovascular disease. We aim to discuss the biomarkers' pathophysiological roles as a reflection of the cardiac remodelling mechanisms in the presence of DM.REVIEW METHODS AND DATA SOURCES: We performed the literature search from June to September 2021 using the following databases: MEDLINE, Scopus, Web of Science, PubMed, and Cochrane library. Articles that met our inclusion criteria were screened and appraised before being included in this review. The PRISMA guidelines for Scoping Reviews were followed.RESULTS: Our literature search identified a total of 43 eligible articles, which were included in this scoping review. We identified 15 different biomarkers that were used to determine signs of cardiac remodelling in cardiovascular disease (CVD) and people with DM. NT-proBNP was identified as the most frequently employed biomarker in this context, however we also identified emerging biomarkers including hs-CRP, hs-cTnT, and Galectin-3.CONCLUSIONS: There is a complex relationship between DM and cardiovascular health, where more research is needed. Current biomarkers reflective of adverse cardiac remodelling in DM are often used to diagnose other CVDs, such as NT-proBNP for heart failure. Hence there is a need for identification of specific biomarkers that can detect early signs of cardiac remodelling. Further research into these biomarkers and mechanisms can deepen our understanding on their role in DM-associated CVD and lead to better preventative therapies.PMID:36782075 | DOI:10.1111/dme.15064

Methods Mol Biol. 2023;2628:489-504. doi: 10.1007/978-1-0716-2978-9_29.ABSTRACTMass spectrometry remains one of the gold standard approaches in examining the lipidome in biological samples. Recently, advancements in chromatography and mass spectrometry approaches have enabled broad coverage of the lipidome. However, many limitations still exist, and lipidomic analysis often requires a fine balance between coverage of the lipidome, structural detail, and sample throughput. For biomedical and clinical research using human samples, the diversity and natural variation between different individuals necessitate larger sample numbers to identify significant associations with clinical outcomes and account for potential confounding factors. Here we describe a targeted lipidomics workflow that enables reproducible profiling of thousands of plasma samples in a systematic manner, while maintaining good structural detail and high coverage of the lipidome.PMID:36781803 | DOI:10.1007/978-1-0716-2978-9_29

Methods Mol Biol. 2023;2628:207-219. doi: 10.1007/978-1-0716-2978-9_14.ABSTRACTIn the last years, platelet concentrates such as leukocyte-platelet-rich fibrin (L-PRF) have been used in different clinical scenarios as a huge source of growth factors to enhance wound healing. However, platelet concentrates release many other proteins that also participate in tissue regeneration processes. In this context, the analysis of the L-PRF secretome would provide relevant information on the different proteins and growth factors released by these platelet concentrates, how such secretion varies with the time, and how relevant this could be for the regenerative properties of these products. In the present chapter, we will provide a protocol for isolation, culture, and secretome analysis of L-PRF membranes. Qualitative and quantitative proteomic approaches will be presented, including gel-based and quantitative Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based approaches. This protocol has been recently applied with success to define the L-PRF secretome composition, setting the stage for further research that can provide relevant information on the clinical properties of these platelet concentrates' subtype.PMID:36781788 | DOI:10.1007/978-1-0716-2978-9_14

Sci Rep. 2023 Feb 13;13(1):2533. doi: 10.1038/s41598-023-28551-x.ABSTRACTTongkat ali commonly known as Malaysian Ginseng (Eurycoma longifolia) is a herbal root worldwide available in nutraceuticals, either as a crude powder or capsules blended with other herbal products. Herein, a multiplexed metabolomics approach based on nuclear magnetic resonance (NMR) and solid-phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS) was applied for authentic tongkat ali extract vs some commercial products quality control analysis. NMR metabolite fingerprinting identified 15 major metabolites mostly ascribed to sugars, organic and fatty acids in addition to quassinoids and cinnamates. Following that, multivariate analysis as the non-supervised principal component analysis (PCA) and supervised orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied revealing that differences were related to fatty acids and 13,21-dihydroeurycomanone being more enriched in authentic root. SPME-GC-MS aroma profiling led to the identification of 59 volatiles belonging mainly to alcohols, aldehydes/furans and sesquiterpene hydrocarbons. Results revealed that aroma of commercial products showed relatively different profiles being rich in vanillin, maltol, and methyl octanoate. Whereas E-cinnamaldehyde, endo-borneol, terpinen-4-ol, and benzaldehyde were more associated to the authentic product. The present study shed the light for the potential of metabolomics in authentication and standardization of tongkat ali and identification of its true flavor composition.PMID:36781893 | DOI:10.1038/s41598-023-28551-x

Nat Commun. 2023 Feb 13;14(1):809. doi: 10.1038/s41467-023-36370-x.ABSTRACTRearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.PMID:36781850 | DOI:10.1038/s41467-023-36370-x

Cell Death Dis. 2023 Feb 13;14(2):116. doi: 10.1038/s41419-023-05629-y.ABSTRACTFKBP51 plays a relevant role in sustaining cancer cells, particularly melanoma. This cochaperone participates in several signaling pathways. FKBP51 forms a complex with Akt and PHLPP, which is reported to dephosphorylate Akt. Given the recent discovery of a spliced FKBP51 isoform, in this paper, we interrogate the canonical and spliced isoforms in regulation of Akt activation. We show that the TPR domain of FKBP51 mediates Akt ubiquitination at K63, which is an essential step for Akt activation. The spliced FKBP51, lacking such domain, cannot link K63-Ub residues to Akt. Unexpectedly, PHLPP silencing does not foster phosphorylation of Akt, and its overexpression even induces phosphorylation of Akt. PHLPP stabilizes levels of E3-ubiquitin ligase TRAF6 and supports K63-ubiquitination of Akt. The interactome profile of FKBP51 from melanoma cells highlights a relevant role for PHLPP in improving oncogenic hallmarks, particularly, cell proliferation.PMID:36781840 | DOI:10.1038/s41419-023-05629-y

Methods Mol Biol. 2023;2628:557-577. doi: 10.1007/978-1-0716-2978-9_32.ABSTRACTIn targeted proteomics experiments, selecting the appropriate proteotypic peptides as surrogate for the target protein is a crucial pre-acquisition step. This step is largely a bioinformatics exercise that involves integrating information on the peptides and proteins and using various software tools and knowledgebases. We present here a few resources that automate and simplify the selection process to a great degree. These tools and knowledgebases were developed primarily to streamline targeted proteomics assay development and include PeptidePicker, PeptidePickerDB, MRMAssayDB, MouseQuaPro, and PeptideTracker. We have used these tools to develop and document thousands of targeted proteomics assays, many of them for plasma proteins with focus on human and mouse. An important aspect in all these resources is the integrative approach on which they are based. Using these tools in the first steps of designing a singleplexed or multiplexed targeted proteomic experiment can reduce the necessary experimental steps tremendously. All the tools and knowledgebases we describe here are Web-based and freely accessible so scientists can query the information conveniently from the browser. This chapter provides an overview of these software tools and knowledgebases, their content, and how to use them for targeted plasma proteomics. We further demonstrate how to use them with the results of the HUPO Human Plasma Proteome Project to produce a new database of 3.8 k targeted assays for known human plasma proteins. Upon experimental validation, these assays should help in the further quantitative characterizing of the plasma proteome.PMID:36781806 | DOI:10.1007/978-1-0716-2978-9_32

Methods Mol Biol. 2023;2628:439-473. doi: 10.1007/978-1-0716-2978-9_27.ABSTRACTPreclinical and clinical trials require rapid, precise, and multiplexed analytical methods to characterize the complex samples and to allow high-throughput biomarker monitoring with low consumption of sample material. Targeted proteomics has been used to address these challenges when quantifying protein abundances in complex biological matrices. In many of these studies, blood plasma is collected either as the main research or diagnostic sample or in combination with other specimens. Mass spectrometry (MS)-based targeted proteomics using multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) with stable isotope-labeled internal standard (SIS) peptides allows robust characterization of blood plasma protein via absolute quantification. Compared to other commonly used technologies like enzyme-linked immunosorbent assay (ELISA), targeted proteomics is faster, more sensitive, and more cost-effective. Here we describe a protocol for the quantification of proteins in blood plasma using targeted MRM proteomics with heavy-labeled internal standards. The 270-protein panel allows rapid and robust absolute quantitative proteomic characterization of blood plasma in a 1 h gradient. The method we describe here works for non-depleted plasma, which makes it simple and easy to implement. Moreover, the protocol works with the two most commonly used blood plasma collection methods used in practice, namely, either K2EDTA or sodium citrate as anticoagulants.PMID:36781801 | DOI:10.1007/978-1-0716-2978-9_27

Methods Mol Biol. 2023;2628:109-125. doi: 10.1007/978-1-0716-2978-9_8.ABSTRACTBlood in the circulatory system carries information of physiological and pathological status of the human body, so blood proteins are often used as biomarkers for diagnosis, prognosis, and therapy. Human blood proteome can be explored by the latest technologies in mass spectrometry (MS), creating an opportunity of discovering new disease biomarkers. The extreme dynamic range of protein concentrations in blood, however, poses a challenge to detect proteins of low abundance, namely, tissue leakage proteins. Here, we describe a strategy to directly analyze undepleted blood samples by extensive liquid chromatography (LC) fractionation and 18-plex tandem-mass-tag (TMT) mass spectrometry. The proteins in blood specimens (e.g., plasma or serum) are isolated by acetone precipitation and digested into peptides. The resulting peptides are TMT-labeled, separated by basic pH reverse-phase (RP) LC into at least 40 fractions, and analyzed by acidic pH RPLC and high-resolution MS/MS, leading to the quantification of ~3000 unique proteins. Further increase of basic pH RPLC fractions and adjustment of the fraction concatenation strategy can enhance the proteomic coverage (up to ~5000 proteins). Finally, the combination of multiple batches of TMT experiments allows the profiling of hundreds of blood samples. This TMT-MS-based method provides a powerful platform for deep proteome profiling of human blood samples.PMID:36781782 | DOI:10.1007/978-1-0716-2978-9_8