Differences In Protein Abundance Research Articles

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding of a subset of proteins with alpha helical domains.

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we revealed the expression of RPL39L protein beyond mouse germ cells, in human pluripotent cells, cancer cell lines and tissue samples. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread autophagy. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

Full Native timsTOF PASEF-Enabled Quantitative Proteomics with the i2MassChroQ Software Package.

Ion mobility mass spectrometry has become popular in proteomics lately, in particular because the Bruker timsTOF instruments have found significant adoption in proteomics facilities. The Bruker's implementation of the ion mobility dimension generates massive amounts of mass spectrometric data that require carefully designed software both to extract meaningful information and to perform processing tasks at reasonable speed. In a historical move, the Bruker company decided to harness the skills of the scientific software development community by releasing to the public the timsTOF data file format specification. As a proteomics facility that has been developing Free Open Source Software (FOSS) solutions since decades, we took advantage of this opportunity to implement the very first FOSS proteomics complete solution to natively read the timsTOF data, low-level process them, and explore them in an integrated quantitative proteomics software environment. We dubbed our software i2MassChroQ because it implements a (peptide)identification-(protein)inference-mass-chromatogram-quantification processing workflow. The software benchmarking results reported in this paper show that i2MassChroQ performed better than competing software on two critical characteristics: (1) feature extraction capability and (2) protein quantitative dynamic range. Altogether, i2MassChroQ yielded better quantified protein numbers, both in a technical replicate MS runs setting and in a differential protein abundance analysis setting.

Quantitative Label-Free Proteomic Analysis of Mouse Ovarian Antral Follicles Following Oral Exposure to a Human Relevant Mixture of Three Phthalates.

Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are used in personal and medical care products. In the ovary, antral follicles are essential for steroidogenesis and ovulation. DBP, BBP, and DEHP are known to inhibit mouse antral follicle growth and ovulation in vitro, and associate with decreased antral follicle counts in women. Given that the in vivo effects of a three-phthalate mixture on antral follicles are unknown, we evaluated the effects of a human relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. Adult CD-1 female mice were fed corn oil (vehicle), or two dose levels of a phthalate mixture based on estimated exposures in general (32 µg/kg/day; PHT 32) and occupationally exposed (500 µg/kg/day; PHT 500) populations for 10 days. Antral follicles (>250 µm) were isolated and subjected to proteome profiling via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were detected, of which 194 were differentially abundant between vehicle and PHT 32, and 136 between vehicle and PHT 500. Bioinformatic analysis revealed significantly different responses between the two phthalate doses. Protein abundance differences in the PHT 32 exposure mapped to cytoplasm, mitochondria, and lipid metabolism; while those in the PHT 500 exposure mapped to cytoplasm, nucleus, and phosphorylation. When both doses altered proteins mapped to common processes, the associated predicted transcription factors were different. These findings provide novel mechanistic insight into phthalate-associated, ovary-driven reproductive outcomes in women.

Decreased Extracellular Vesicle Vasorin in Severe Preeclampsia Plasma Mediates Endothelial Dysfunction.

Preeclampsia (PE) is a serious pregnancy complication affecting 5-8% of pregnancies globally. It is a leading cause of maternal and neonatal morbidity and mortality. Despite its prevalence, the underlying mechanisms of PE remain unclear. This study aimed to determine the potential role of vasorin (VASN) in PE pathogenesis by investigating its levels in extracellular vesicles (EV) and its effects on vascular function. We conducted unbiased proteomics on urine-derived EV from severe PE (sPE) and normotensive pregnant women (NTP), identifying differential protein abundances. Out of one hundred and twenty proteins with ≥ ±1.5-fold regulation at P<0.05 between sPE and NTP, we focused on Vasorin (VASN), which is downregulated in sPE in urinary EV, in plasma EV and in the placenta and is a known regulator of vascular function. We generated EV with high VASN content from both human and murine placenta explants (Plex EV), which recapitulated disease-state-dependent effects on vascular function observed when treating murine aorta rings (MAR) or human aortic endothelial cells (HAEC) with murine or human plasma-derived EV. In normal murine pregnancy, VASN increases with gestational age (GA), and VASN is decreased in plasma EV, in placenta tissue and in Plex EV after intravenous administration of adenovirus encoding short FMS-like tyrosine kinase 1 (sFLT-1), a murine model of PE (murine-PE). VASN is decreased in plasma EV, in placenta tissue and in EV isolated from conditioned media collected from placenta explants (Plex EV) in patients with sPE as compared to NTP. Human sPE and murine-PE plasma EV and Plex EV impair migration, tube formation, and induces apoptosis in human aortic endothelial cells (HAEC) and inhibit acetylcholine-induced vasorelaxation in murine vascular rings (MAR). VASN over-expression counteracts the effects of sPE EV treatment in HAEC and MAR. RNA sequencing revealed that over-expression or knock down of VASN in HAEC results in contrasting effects on transcript levels of hundreds of genes associated with vasculogenesis, endothelial cell proliferation, migration and apoptosis. The data suggest that VASN, delivered to the endothelium via EV, regulates vascular function and that the loss of EV VASN may be one of the mechanistic drivers of PE. What is NewVASN in circulating plasma EV in sPE is reduced compared with VASN content in plasma EV of gestational age-matched pregnant women.VASN is encapsulated and transported in EV and plays a pro-angiogenic role during pregnancy.VASN should be explored both for its pro-angiogenic mechanistic role and as a novel biomarker and potential predictive diagnostic marker for the onset and severity of PE.What Are the Clinical Implications?VASN plays a role in maintaining vascular health and the normal adaptive cardiovascular response in pregnancy. A decrease of VASN is observed in sPE patients contributing to cardiovascular maladaptation.Strategies to boost diminished VASN levels and/or to pharmacologically manipulate mechanisms downstream of VASN may be explored for potential therapeutic benefit in PE.The decrease in EV-associated VASN could potentially be used as a (predictive) biomarker for PE.

Unbiased clustering and molecular characterisation of novel metabolic phenotypes in a heart failure cohort

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DIASyM cluster Heart Failure is a heterogeneous clinical syndrome characterized by the inability to pump enough blood and thus provide enough oxygen to the human body. The current classification of heart failure is based on the left ventricular ejection faction (LVEF). Although the classification clearly defines heart failure subtypes and stages, and clinical guidelines are available for their management, it is preferable to prevent the disorder or take control over it from its onset. Evidence suggests strong molecular connections between human metabolism and heart failure. Exploiting the unique and diverse molecular characteristics that drive heart failure could be an effective strategy for patient stratification, to identify new therapeutic targets and to personalize treatments. The current research attempts to identify clinically relevant metabolic subgroups of individuals with the help of a similarity network-based approach, based on targeted proteomics data of a heart failure cohort. With Olink targeted proteomics technology, six protein abundance panels (547 distinct proteins) from 3063 subjects (1103 females) were obtained. Using nonlinear combination methods, the created networks were integrated into a single similarity network and then used for clustering with unbiased network-based approaches. The results have distinguished the subgroups with worse outcome prediction, supported by significantly different clinical profiles. Surprisingly, the significant clinical parameters have shown the dominance of not only classical metabolic signatures (diabetes) but also parameters related to kidney and liver function. The differential protein abundance profiles were then compared among clusters to derive novel insights into clinical subtypes, and have highlighted a significant contribution of inflammatory proteins to the unbiased subtyping rationale. The study attempted to improve previous patient stratification efforts, by having a large sample size with a broader variety of molecular variables. These results will pave the way to precision medicine, and will allow indirectly to integrate and/or adjust the analysis by adding clinically relevant social parameters such as lifestyle, parents’ anamnesis, demographics that are often left out due to their lower contribution at the pre-processing steps in comparison to more objective laboratory parameters. Previous studies have already clustered the heart failure population, based on various clinical and biochemical parameters. However, the clustering was guided by well-established metabolic parameters and thus, the results can be highly biased. The current project allows an unbiased, data-driven approach that holds more potential to identify novel and clinically relevant metabolic subgroups and to define new molecular targets.

Remodeling of the Human Skeletal Muscle Proteome Found After Long-Term Endurance Training but Not After Strength Training

Regular exercise training has tremendous systemic tissue-specific health benefits, but the molecular adaptations to long-term exercise training are not completely understood. The aim of the current study was to investigate the skeletal muscle proteome of highly endurance-trained, strength-trained and untrained individuals and performed exercise- and sex-specific analyses. As a previous investigation from our lab showed limited baseline differences at the gene expression level between highly strength-trained males and untrained males, we hypothesized that the that long-term resistance training results in baseline differences in protein abundances. Additionally, we hypothesized that endurance-trained individuals would have a greater abundance of mitochondrial proteins compared with the untrained and the strength-trained individuals. Liquid-chromatography followed by mass-spectrometry (LC-MS/MS) was performed on skeletal muscle tissue from all individuals. A total of 6713 proteins were detected, with 4269 proteins being expressed in at least 50% of the samples. Over 650 proteins were differentially expressed in the skeletal muscle of endurance-trained individuals compared with controls. Strikingly, 92% of the shared proteins with higher expression in both the male and female endurance groups were known mitochondrial proteins. In contrast to the findings in endurance trained individuals, minimal differences were found in strength-trained individuals and between females and males. Lastly, to determine the preventive-medicine capabilities of long-term training, a co-expression network and comparative literature analysis revealed key proteins and pathways related to the health benefits of exercise, which were primarily related to differences in mitochondrial protein abundance. This network is available as an interactive database resource ( https://inetmodels.com ) where investigators can correlate clinical data with global gene and protein expression data for hypothesis generation. In conclusion, we suggest that long-term endurance training but not strength training results in a r emodeling of the skeletal muscle proteome in humans. This work was financially supported by grants from the Swedish Research Council (2018-02932), the Swedish Center for Sports Research (2020-D0007, P2022-0082), the National Science Foundation (Award Number: 1951792), the Whitaker International Program and the Karolinska Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unraveling the Mechanisms of Efficient Phosphorus Utilization in Popcorn (Zea mays L. var. everta): Insights from Proteomic and Metabolite Analysis.

The expansion of agriculture and the need for sustainable practices drives breeders to develop plant varieties better adapted to abiotic stress such as nutrient deficiency, which negatively impacts yields. Phosphorus (P) is crucial for photosynthesis and plant growth, but its availability in the soil is often limited, hampering crop development. In this study, we examined the response of two popcorn inbred lines, L80 and P7, which have been characterized previously as P-use inefficient and P-use efficient, respectively, under low (stress) and high P (control) availability. Physiological measurements, proteomic analysis, and metabolite assays were performed to unravel the physiological and molecular responses associated with the efficient use of P in popcorn. We observed significant differences in protein abundances in response to the P supply between the two inbred lines. A total of 421 differentially expressed proteins (DEPs) were observed in L80 and 436 DEPs in P7. These proteins were involved in photosynthesis, protein biosynthesis, biosynthesis of secondary metabolites, and energy metabolism. In addition, flavonoids accumulated in higher abundance in P7. Our results help us understand the major components of P utilization in popcorn, providing new insights for popcorn molecular breeding programs.

Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SignificanceCiliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Multi-Omics Profiling Reveals Phenotypic and Functional Heterogeneity of Neutrophils in COVID-19.

Accumulating evidence has revealed unexpected phenotypic heterogeneity and diverse functions of neutrophils in several diseases. Coronavirus disease (COVID-19) can alter the leukocyte phenotype based on disease severity, including neutrophil activation in severe cases. However, the plasticity of neutrophil phenotypes and their relative impact on COVID-19 pathogenesis has not been well addressed. This study aimed to identify and validate the heterogeneity of neutrophils in COVID-19 and evaluate the functions of each subpopulation. We analyzed public single-cell RNA-seq, bulk RNA-seq, and proteome data from healthy donors and patients with COVID-19 to investigate neutrophil subpopulations and their response to disease pathogenesis. We identified eight neutrophil subtypes: pro-neutrophil, pre-neutrophil, immature neutrophil, and five mature neutrophil subpopulations. The subtypes exhibited distinct features, including diverse activation signatures and multiple enriched pathways. The pro-neutrophil subtype was associated with severe and fatal disease, while the pre-neutrophil subtype was particularly abundant in mild/moderate disease. One of the mature neutrophil subtypes showed consistently large fractions in patients with different disease severity. Bulk RNA-seq dataset analyses using a cellular deconvolution approach validated the relative abundances of neutrophil subtypes and the expansion of pro-neutrophils in severe COVID-19 patients. Cell-cell communication analysis revealed representative ligand-receptor interactions among the identified neutrophil subtypes. Further investigation into transcription factors and differential protein abundance revealed the regulatory network differences between healthy donors and patients with severe COVID-19. Overall, we demonstrated the complex interactions among heterogeneous neutrophil subtypes and other blood cell types during COVID-19 disease. Our work has great value in terms of both clinical and public health as it furthers our understanding of the phenotypic and functional heterogeneity of neutrophils and other cell populations in multiple diseases.

Discriminatory power of MALDI-TOF MS protein profiling analysis of pork meat and meat products

Forty different sample preparation methods were tested to obtain the most informative MALDI-TOF MS protein profiles of pork meat. Extraction by 25% formic acid with the assistance of zirconia-silica beads followed by defatting by methanol:chloroform mixture (1:1, v/v) and deposition by using the layer-by-layer method was determined as the optimum sample preparation protocol. The discriminatory power of the method was then examined on samples of pork meat and meat products. The method was able to discriminate between selected salami based on the production method and brand and was able to monitor the ripening process in salami. However, it was not able to differentiate between different brands of pork ham or closely located parts of pork meat. In the latter case, a more comprehensive analysis using LC-MS/MS was used to assess the differences in protein abundance and their relation to the outputs of MALDI – TOF MS profiling.

Abstract 3867: Identification of novel biomarkers of immune toxicity from CAR T-cell therapy using ultrasensitive NULISA™ proteome technology

Abstract Introduction: The efficacy of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in patients with relapsed/refractory large B-cell lymphoma (LBCL) is limited by acute toxic events, most notably cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Identification of biomarkers of toxicities can allow for selection of high-risk patients and elucidate targetable mechanisms for mitigation. Previous biomarker studies have been constrained by relatively narrow panels of potential mediators and limited time points. Here, we employed NULISA, a novel ultrasensitive assay capable of simultaneously quantifying 204 inflammatory proteins from a single sample, to identify temporal proteome associations with acute toxicities in anti-CD19 CAR T-cell treated patients. Methods: Baseline and post-treatment peripheral blood samples from 80 patients with LBCL who underwent anti-CD19 CAR-T cell therapy were collected within specific time intervals. Plasma samples (n=480) were analyzed with NULISA. CRS and ICANS were graded according to ASTCT consensus criteria. Patients were grouped according to their maximum CRS and ICANS scores. Differential protein abundance across the toxicity groups was assessed using linear mixed effects models fit for each protein, including a time by toxicity group interaction. Proteins showing significant associations were subjected to pathway enrichment and network analysis. Results: Severe CRS and ICANS patient groups showed proteome upregulation beginning on day 1-2 and peaking at day 6-9, followed by extensive downregulation on day 11-16. The strongest upregulated pathway associations of severe CRS and ICANS were inflammatory response, IL-17 signaling, non-genomic action of vitamin D3, regulation of leukocyte proliferation, and cellular extravasation. Downregulated pathways included anti-microbial humoral response and TNFs binding to receptors. In addition to the previously identified inflammatory proteins IL-6, IFNγ, sIL-2Rα, CXCL8, CCL2, our analysis revealed significant association of several novel immune regulators and mediators. These included upregulated Th2 cytokines, IL-17A, IL-22, GZMB, CTLA4, IFNA1;IFNA13, and downregulated S100A12, IL-12B, CCL22, BDNF and TNFSFs, revealing exquisite temporal orchestration of lymphoid and myeloid activities during CRS and ICANS development. Conclusions: This study represents the most comprehensive characterization of immune response to CAR T-cell therapy to date. Using the novel NULISA technology, we identified new proteins and functional pathways associated with CAR T-cell-induced toxicity, implicating them as potential biomarkers. These previously unidentified factors also provide a platform to further investigate the causative immune mechanisms of acute toxicities in CAR-T cell therapy. Citation Format: Riley Kirkpatrick, Joanne Beer, Manishkumar S. Patel, Akansha Jalota, Agrima Mian, Ishara S. Ariyapala, Qinyu Hao, Wei Feng, Xiao-Jun Ma, Yuling Luo, Brian T. Hill, Neetu Gupta. Identification of novel biomarkers of immune toxicity from CAR T-cell therapy using ultrasensitive NULISA™ proteome technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3867.

Histological and molecular difference in albumen quality between post-adolescent hens and aged hens

The decline in albumen quality resulting from aging hens poses a threat to the financial benefits of the egg industry. Exploring the underlying mechanisms from the perspective of cell molecules of albumen formation is significant for the efficient regulation of albumen quality. Two individual groups of Hy-Line Brown layers with ages of 40 (W40) and 100 (W100) wk old were used in the present study. Each group contained over 2,000 birds. This study assessed the egg quality, biochemical indicators and physiological status of hens between W40 and W100. Subsequently, a quantitative proteomic analysis was conducted to identify differences in protein abundance in magnum tissues between W40 and W100. In the W40 group, significant increases (P < 0.05) were notable for albumen quality (thick albumen solid content, albumen height, Haugh unit), serum indices (calcium, estrogen, and progesterone levels), magnum histomorphology (myosin light-chain kinase content, secretory capacity, mucosal fold, goblet cell count and proportion) as well as the total antioxidant capacity of the liver. However, the luminal diameter of the magnum, albumen gel properties and random coil of the albumen were increased (P < 0.05) in the W100 group. The activity of glutathione, superoxidase dismutase, and malondialdehyde in the liver, magnum, and serum did not vary (P > 0.05) among the groups. Proteomic analysis revealed the identification of 118 differentially expressed proteins between the groups, which comprised proteins associated with protein secretion, DNA damage and repair, cell proliferation, growth, antioxidants, and apoptosis. Furthermore, Kyoto Encyclopedia of Genes pathway analysis revealed that BRCA2 and FBN1 were significantly downregulated in Fanconi anemia (FA) and TGF-β signaling pathways in W100, validated through quantitative real-time PCR (qRT-PCR). In conclusion, significant age-related variations in albumen quality, and magnum morphology are regulated by proteins involved in antioxidant capacity.

A magnetic separation method for isolating and characterizing the biomolecular corona of lipid nanoparticles

Lipid nanoparticle (LNP) formulations are a proven method for the delivery of nucleic acids for gene therapy as exemplified by the worldwide rollout of LNP-based RNAi therapeutics and mRNA vaccines. However, targeting specific tissues or cells is still a major challenge. After LNP administration, LNPs interact with biological fluids (i.e., blood), components of which adsorb onto the LNP surface forming a layer of biomolecules termed the "biomolecular corona (BMC)" which affects LNP stability, biodistribution, and tissue tropism. The mechanisms by which the BMC influences tissue- and cell-specific targeting remains largely unknown, due to the technical challenges in isolating LNPs and their corona from complex biological media. In this study, we present a new technique that utilizes magnetic LNPs to isolate LNP-corona complexes from unbound proteins present in human serum. First, we developed a magnetic LNP formulation, containing >40 superparamagnetic iron oxide nanoparticles (IONPs)/LNP, the resulting LNPs containing iron oxide nanoparticles (IOLNPs) displayed a similar particle size and morphology as LNPs loaded with nucleic acids. We further demonstrated the isolation of the IOLNPs and their corresponding BMC from unbound proteins using a magnetic separation (MS) system. The BMC profile of LNP from the MS system was compared to size exclusion column chromatography and further analyzed via mass spectrometry, revealing differences in protein abundances. This new approach enabled a mild and versatile isolation of LNPs and its corona, while maintaining its structural integrity. The identification of the BMC associated with an intact LNP provides further insight into LNP interactions with biological fluids.

Proteomic Analysis of a Rat Streptozotocin Model Shows Dysregulated Biological Pathways Implicated in Alzheimer's Disease.

Alzheimer's Disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory loss and cognitive impairment, affecting 35 million individuals worldwide. Intracerebroventricular (ICV) injection of low to moderate doses of streptozotocin (STZ) in adult male Wistar rats can reproduce classical physiopathological hallmarks of AD. This biological model is known as ICV-STZ. Most studies are focused on the description of behavioral and morphological aspects of the ICV-STZ model. However, knowledge regarding the molecular aspects of the ICV-STZ model is still incipient. Therefore, this work is a first attempt to provide a wide proteome description of the ICV-STZ model based on mass spectrometry (MS). To achieve that, samples from the pre-frontal cortex (PFC) and hippocampus (HPC) of the ICV-STZ model and control (wild-type) were used. Differential protein abundance, pathway, and network analysis were performed based on the protein identification and quantification of the samples. Our analysis revealed dysregulated biological pathways implicated in the early stages of late-onset Alzheimer's disease (LOAD), based on differentially abundant proteins (DAPs). Some of these DAPs had their mRNA expression further investigated through qRT-PCR. Our results shed light on the AD onset and demonstrate the ICV-STZ as a valid model for LOAD proteome description.

Two-stage association study of mitochondrial DNA variants in allergic rhinitis

BackgroundCorrelations between mitochondrial DNA (mtDNA) and allergic rhinitis (AR) have not been reported before. This study aimed to better understand the mitochondrial genome profile with AR and to investigate the associations between AR in China and the mitochondrial genome at a single variant and gene level.MethodsMitochondrial sequencing was conducted on a total of 134 unrelated individual subjects (68 patients with AR, 66 healthy controls) at discovery stage. Heteroplasmy was analyzed using the Mann-Whitney U test. Sequence kernel association tests (SKAT) were conducted to study the association between mitochondrial genes and AR. Single-variant analysis was performed using logistic regression analysis and further validated in 120 subjects (69 patients with AR, 51 healthy controls). Candidate genes were further explored based on differences in mRNA and protein abundance in nasal mucosal tissue.ResultsIn the discovery stage, 886 variants, including 836 SNV and 50 indels, were identified with mitochondrial sequencing. No statistically significant differences were identified for the mitochondrial heteroplasmy or SKAT analysis between these two groups after applying a Boferroni correction. One nonsynonymous variants, rs3135028 (MT8584.G/A) in ATP6, was related to a reduced risk of AR in both the discovery and validation cohorts. Furthermore, mRNA levels of MT-ATP6 in nasal mucosal tissue were significantly lower in AR individuals than in controls (P < 0.05).ConclusionsIn a two-stage analysis of associations between AR and mtDNA variations, mitochondrial gene maps of Chinese patients with AR indicated that the ATP6 gene was probably associated with AR at the single-variant level.

Evolved changes in phenotype across skeletal muscles in deer mice native to high altitude.

The cold and hypoxic conditions at high altitude necessitate high metabolic O2 demands to support thermogenesis while hypoxia reduces O2 availability. Skeletal muscles play key roles in thermogenesis, but our appreciation of muscle plasticity and adaptation at high altitude has been hindered by past emphasis on only a small number of muscles. We examined this issue in deer mice (Peromyscus maniculatus). Mice derived from both high-altitude and low-altitude populations were born and raised in captivity and then acclimated as adults to normoxia or hypobaric hypoxia (12 kPa O2 for 6-8 wk). Maximal activities of citrate synthase (CS), cytochrome c oxidase (COX), β-hydroxyacyl-CoA dehydrogenase (HOAD), hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) were measured in 20 muscles involved in shivering, locomotion, body posture, ventilation, and mastication. Principal components analysis revealed an overall difference in muscle phenotype between populations but no effect of hypoxia acclimation. High-altitude mice had greater activities of mitochondrial enzymes and/or lower activities of PK or LDH across many (but not all) respiratory, limb, core and mastication muscles compared with low-altitude mice. In contrast, chronic hypoxia had very few effects across muscles. Further examination of CS in the gastrocnemius showed that population differences in enzyme activity stemmed from differences in protein abundance and mRNA expression but not from population differences in CS amino acid sequence. Overall, our results suggest that evolved increases in oxidative capacity across many skeletal muscles, at least partially driven by differences in transcriptional regulation, may contribute to high-altitude adaptation in deer mice.NEW & NOTEWORTHY Most previous studies of muscle plasticity and adaptation in high-altitude environments have focused on a very limited number of skeletal muscles. Comparing high-altitude versus low-altitude populations of deer mice, we show that a large number of muscles involved in shivering, locomotion, body posture, ventilation, and mastication exhibit greater mitochondrial enzyme activities in the high-altitude population. Therefore, evolved increases in mitochondrial oxidative capacity across skeletal muscles contribute to high-altitude adaptation.

Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. Sex- and strain-specific differences in protein abundances are identified and described, and the measured values are freely accessible via our MouseQuaPro database: http://mousequapro.proteincentre.com . Together, this large library of quantitative MRM-MS assays established in mice and the measured baseline protein abundances represent an important resource for research involving mouse models.

Proteomic Profiles of Whole Seeds, Hulls, and Dehulled Seeds of Two Industrial Hemp (Cannabis sativa L.) Cultivars.

As a source of nutritionally important components, hemp seeds are often dehulled for consumption and food applications by removing the hard hulls, which increases their nutritional value. The hulls thus become waste, although they may contain valuable protein items, about which there is a lack of information. The present work is therefore aimed at evaluating the proteome of hemp (Cannabis sativa L.) at the whole-seed, dehulled seed, and hull levels. The evaluation was performed on two cultivars, Santhica 27 and Uso-31, using LC-MS/MS analysis. In total, 2833 protein groups (PGs) were identified, and their relative abundances were determined. A set of 88 PGs whose abundance exceeded 1000 ppm (MP88 set) was considered for further evaluation. The PGs of the MP88 set were divided into ten protein classes. Seed storage proteins were found to be the most abundant protein class: the averages of the cultivars were 65.5%, 71.3%, and 57.5% for whole seeds, dehulled seeds, and hulls, respectively. In particular, 11S globulins representing edestin (three PGs) were found, followed by 7S vicilin-like proteins (four PGs) and 2S albumins (two PGs). The storage 11S globulins in Santhica 27 and Uso-31 were found to have a higher relative abundance in the dehulled seed proteome (summing to 58.6 and 63.2%) than in the hull proteome (50.5 and 54%), respectively. The second most abundant class of proteins was oleosins, which are part of oil-body membranes. PGs belonging to metabolic proteins (e.g., energy metabolism, nucleic acid metabolism, and protein synthesis) and proteins related to the defence and stress responses were more abundant in the hulls than in the dehulled seeds. The hulls can, therefore, be an essential source of proteins, especially for medical and biotechnological applications. Proteomic analysis has proven to be a valuable tool for studying differences in the relative abundance of proteins between dehulled hemp seeds and their hulls among different cultivars.

Tubular deficiency of ABCA1 augments cholesterol- and Na+-dependent effects on systemic blood pressure in male mice.

Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na+ channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na+-dependent changes in blood pressure (BP). Transgenic mice (TgPAX8rtTA;tetO-Cre/+) expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; n = 10, P < 0.05) compared with wild-type littermates (WT; n = 9). Tail-cuff SBP of ABCA1FF (n = 11) was not only greater post dox, but also during cholesterol or high Na+ feeding (P < 0.05) compared with WT mice (n = 15). A Na+-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding (P < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF (n = 7; 45.2 ± 8.7 µL/g body wt) versus WT (n = 7; 33.1 ± 6.9 µL/g body wt; P < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na+ sensitivity of SBP, which we speculate is partly related to Na+ transporters and channels.NEW & NOTEWORTHY Cholesterol has been linked to greater Na+ channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.

Systematic profiling of ale yeast protein dynamics across fermentation and repitching.

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is among the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout 2 fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.