Differences In Protein Abundance Research Articles

Dynamic thresholding and tissue dissociation optimization for CITE-seq identifies differential surface protein abundance in metastatic melanoma

Multi-omics profiling by CITE-seq bridges the RNA-protein gap in single-cell analysis but has been largely applied to liquid biopsies. Applying CITE-seq to clinically relevant solid biopsies to characterize healthy tissue and the tumor microenvironment is an essential next step in single-cell translational studies. In this study, gating of cell populations based on their transcriptome signatures for use in cell type-specific ridge plots allowed identification of positive antibody signals and setting of manual thresholds. Next, we compare five skin dissociation protocols by taking into account dissociation efficiency, captured cell type heterogeneity and recovered surface proteome. To assess the effect of enzymatic digestion on transcriptome and epitope expression in immune cell populations, we analyze peripheral blood mononuclear cells (PBMCs) with and without dissociation. To further assess the RNA-protein gap, RNA-protein we perform codetection and correlation analyses on thresholded protein values. Finally, in a proof-of-concept study, using protein abundance analysis on selected surface markers in a cohort of healthy skin, primary, and metastatic melanoma we identify CD56 surface marker expression on metastatic melanoma cells, which was further confirmed by multiplex immunohistochemistry. This work provides practical guidelines for processing and analysis of clinically relevant solid tissue biopsies for biomarker discovery.

Statistical Detection of Differentially Abundant Proteins in Experiments with Repeated Measures Designs and Isobaric Labeling.

Repeated measures experimental designs, which quantify proteins in biological subjects repeatedly over multiple experimental conditions or times, are commonly used in mass spectrometry-based proteomics. Such designs distinguish the biological variation within and between the subjects and increase the statistical power of detecting within-subject changes in protein abundance. Meanwhile, proteomics experiments increasingly incorporate tandem mass tag (TMT) labeling, a multiplexing strategy that gains both relative protein quantification accuracy and sample throughput. However, combining repeated measures and TMT multiplexing in a large-scale investigation presents statistical challenges due to unique interplays of between-mixture, within-mixture, between-subject, and within-subject variation. This manuscript proposes a family of linear mixed-effects models for differential analysis of proteomics experiments with repeated measures and TMT multiplexing. These models decompose the variation in the data into the contributions from its sources as appropriate for the specifics of each experiment, enable statistical inference of differential protein abundance, and recognize a difference in the uncertainty of between-subject versus within-subject comparisons. The proposed family of models is implemented in the R/Bioconductor package MSstatsTMT v2.2.0. Evaluations of four simulated datasets and four investigations answering diverse biological questions demonstrated the value of this approach as compared to the existing general-purpose approaches and implementations.

Targeting mitochondria in the aged cerebral vasculature with SS-31, a proteomic study of brain microvessels

Cognitive impairment and dementias during aging such as Alzheimer’s disease are linked to functional decline and structural alterations of the brain microvasculature. Although mechanisms leading to microvascular changes during aging are not clear, loss of mitochondria, and reduced efficiency of remaining mitochondria appear to play a major role. Pharmacological agents, such as SS-31, which target mitochondria have been shown to be effective during aging and diseases; however, the benefit to mitochondrial- and non-mitochondrial proteins in the brain microvasculature has not been examined. We tested whether attenuation of aging-associated changes in the brain microvascular proteome via targeting mitochondria represents a therapeutic option for the aging brain. We used aged male (> 18 months) C57Bl6/J mice treated with a mitochondria-targeted tetrapeptide, SS-31, or vehicle saline. Cerebral blood flow (CBF) was determined using laser speckle imaging during a 2-week treatment period. Then, isolated cortical microvessels (MVs) composed of end arterioles, capillaries, and venules were used for Orbitrap Eclipse Tribrid mass spectrometry. CBF was similar among the groups, whereas bioinformatic analysis revealed substantial differences in protein abundance of cortical MVs between SS-31 and vehicle. We identified 6267 proteins, of which 12% were mitochondria-associated. Of this 12%, 107 were significantly differentially expressed and were associated with oxidative phosphorylation, metabolism, the antioxidant defense system, or mitochondrial dynamics. Administration of SS-31 affected many non-mitochondrial proteins. Our findings suggest that mitochondria in the microvasculature represent a therapeutic target in the aging brain, and widespread changes in the proteome may underlie the rejuvenating actions of SS-31 in aging.

Serum proteome profiles in patients treated with targeted temperature management after out-of-hospital cardiac arrest

BackgroundDefinition of temporal serum proteome profiles after out-of-hospital cardiac arrest may identify biological processes associated with severe hypoxia–ischaemia and reperfusion. It may further explore intervention effects for new mechanistic insights, identify candidate prognostic protein biomarkers and potential therapeutic targets. This pilot study aimed to investigate serum proteome profiles from unconscious patients admitted to hospital after out-of-hospital cardiac arrest according to temperature treatment and neurological outcome.MethodsSerum samples at 24, 48, and 72 h after cardiac arrest at three centres included in the Target Temperature Management after out-of-hospital cardiac arrest trial underwent data-independent acquisition mass spectrometry analysis (DIA-MS) to find changes in serum protein concentrations associated with neurological outcome at 6-month follow-up and targeted temperature management (TTM) at 33 °C as compared to 36 °C. Neurological outcome was defined according to Cerebral Performance Category (CPC) scale as “good” (CPC 1–2, good cerebral performance or moderate disability) or “poor” (CPC 3–5, severe disability, unresponsive wakefulness syndrome, or death).ResultsOf 78 included patients [mean age 66 ± 12 years, 62 (80.0%) male], 37 (47.4%) were randomised to TTM at 36 °C. Six-month outcome was poor in 47 (60.3%) patients. The DIA-MS analysis identified and quantified 403 unique human proteins. Differential protein abundance testing comparing poor to good outcome showed 19 elevated proteins in patients with poor outcome (log2-fold change (FC) range 0.28–1.17) and 16 reduced proteins (log2(FC) between − 0.22 and − 0.68), involved in inflammatory/immune responses and apoptotic signalling pathways for poor outcome and proteolysis for good outcome. Analysis according to level of TTM showed a significant protein abundance difference for six proteins [five elevated proteins in TTM 36 °C (log2(FC) between 0.33 and 0.88), one reduced protein (log2(FC) − 0.6)] mainly involved in inflammatory/immune responses only at 48 h after cardiac arrest.ConclusionsSerum proteome profiling revealed an increase in inflammatory/immune responses and apoptosis in patients with poor outcome. In patients with good outcome, an increase in proteolysis was observed, whereas TTM-level only had a modest effect on the proteome profiles. Further validation of the differentially abundant proteins in response to neurological outcome is necessary to validate novel biomarker candidates that may predict prognosis after cardiac arrest.

Quantitative proteomics and phosphoproteomics reveal insights into mechanisms of ocnus function in Drosophila testis development

BackgroundTestis is the only organ supporting sperm production and with the largest number of proteins and tissue-specific proteins in animals. In our previous studies, we have found that knockdown of ocnus (ocn), a testis-specific gene, resulted in much smaller testis with no germ cells in Drosophila melanogaster. However, the molecular consequences of ocn knockdown in fly testes are unknown.ResultsIn this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions after ocn knockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis of D. melanogaster. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways.ConclusionsGiven the drastic effect of the ocn knockdown on tissue development and testis cells composition, the differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, our results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans.

Comparative proteomic analysis of glomerular proteins in IgA nephropathy and IgA vasculitis with nephritis

BackgroundIgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN.MethodsWe used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0–8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2–44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed.ResultsMore than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-β inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings.ConclusionsThe present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.

Renal mitochondrial metabolism is differentially regulated in male and female kidneys in Ang II -- induced hypertension

Introduction. In 2020, more than 670,000 deaths in the United States had hypertension as a primary or contributing cause. Females generally exhibit less susceptibility to hypertensive renal damage compared to males. It is established that renal mitochondrial dysfunction and associated oxidative stress contribute to hypertension development. Our previous studies have shown that healthy renal mitochondria in the absence of hypertension exhibit sex differences in antioxidant protein abundance, ROS production, and oxygen consumption rates. We hypothesized here that in hypertensive state mitochondrial metabolism in male and female kidneys differentially regulates tissue damage and ultimately hypertension in females. Methods. To address our hypothesis, we used 11-week-old male and female Sprague Dawley rats infused with Angiotensin II (400 ng/kg/min to induce hypertension) or vehicle for 3 weeks via a s.c. osmotic pump. Mitochondria were isolated from the renal cortex and medulla of these rats; mitochondrial membrane potential and H 2 O 2 production were measured by spectrofluorimetry using TMRM and Amplex Red dyes. Two-way ANOVA was utilized for statistical analysis in OriginPro. Metabolic profiles of renal cortices and medullae were generated using UHPLC-HRMS, and metabolites were identified by retention time exact mass using MAVEN and MetaboAnalyst software. Results. Spectrofluorimetry showed decreased membrane potential in both male and female renal medulla in animals infused with Ang II compared to vehicle (p&lt;0.001), while in cortex this difference was only recorded in females (p=0.03). Ang II-infusion reduced H 2 O 2 production in the renal cortex and medulla of female rats (p&lt;0.001) compared to sham groups, while this was not observed in males. In both Ang II and vehicle-infused groups, females maintained higher H 2 O 2 production compared to males. UHPLC-HRMS identified 147 mitochondria-related metabolites in each group. Ang II infusion increased TCA cycle and purine metabolites as well as amino acids in the cortex of both sexes and in male medulla, whereas these changes were not revealed in the female medulla. Interestingly, females exhibited a decrease in amino acid metabolism in the renal medulla. In contrast to the male cortex, the female cortex is responding to oxidative stress induced by hypertension by altering arginine biosynthesis and metabolism. Arginine is a biosynthetic precursor to putrescine, which is involved in polyamine biosynthesis. Conclusions. Male and female kidneys employ divergent metabolic mitochondria-related pathways in hypertensive state. The male cortex may rely more on protein degradation to produce uric acid, an antioxidant, while the female cortex utilizes protein degradation and polyamine biosynthesis in oxidative stress management. R01HL148114 This is the full abstract presented at the American Physiology Summit 2023 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.

Abstract LB005: Insights of renal cell carcinoma from multiomic perspective

Abstract Introduction: Clear cell renal carcinoma is the most frequent form of renal malignancy, with an increasing incidence rate worldwide. In this study we used a multi-omic approach to differentiate normal and tumor tissues in clear cell Renal Cell Carcinoma (ccRCC). Methods: Using transcriptomic data of patients with malignant and adjacent normal tissue samples from gene chip and RNA-Seq cohorts, we identified the top genes over-expressed in ccRCC. We collected surgically resected ccRCC specimens to further investigate the transcriptomic results on the proteome level. The differential protein abundance was evaluated using targeted mass spectrometry (MS). Results: We assembled a database of more than 600 renal tissue samples from NCBI GEO and TCGA and used these to uncover the top genes with higher expression in ccRCC. For protein level analysis 162 malignant and normal kidney tissue samples have been acquired. The most consistently upregulated genes were IGFBP3, PLIN2, PLOD2, PFKP, VEGFA, and CCND1 (p&amp;lt;1E-05 for each gene). Mass spectrometry further validated the differential protein abundance of these genes (IGFBP3, p = 7.53E-18; PLIN2, p = 3.9E-39; PLOD2, p = 6.51E-36; PFKP, p = 1.01E-47; VEGFA, p = 1.40E-22; CCND1, p= 1.04E-24). We also identified proteins correlating with overall survival. Conclusions: We used transcriptomic and proteomic data to identify a minimal panel of proteins highly specific for clear cell renal carcinoma tissues. The introduced gene panel could be used as a promising tool in the clinical setting. Citation Format: Aron Bartha, Zsuzsanna Darula, Gyongyi Munkacsy, Eva Klement, David Fenyo, Peter Nyirady, Balazs Gyorffy. Insights of renal cell carcinoma from multiomic perspective [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB005.

312 Neutrophil Extracellular Trap (NET) Proteins Enriched in Emboli Retrieved from Stroke Patients with Atrial Fibrillation

INTRODUCTION: Recent literature has demonstrated remarkable heterogeneity in the composition of acute ischemic stroke (AIS) emboli from different etiologies, which may directly impact their susceptibility to current standard-of-care therapies. METHODS: The full proteomic composition of retrieved thromboembolic material from 24 patients with AIS was evaluated by mass spectrometry. Of the 1797 total proteins identified, 88 known marker proteins were sorted into groups representing broad classes of embolus components: RBCs, PLTs, PMNs, histones, complement proteins, and other clotting-associated proteins (e.g., fibrinogen). The abundance of individual proteins in patients with AF was compared to that of patients without AF using Mann-Whitney U-tests, with significant differences described by the fold change (FC) in abundance. Functional implications of identified differences were explored using the Gene Ontology (GO) enRIchment anaLysis and visuaLizAtion tool (GOrilla). RESULTS: There were no significant differences in protein abundance in the RBC, complement, or clotting-associated protein groups. One out of the 11 PLT group proteins (p = 0.042, FC = 2.46), eight out of the 15 PMN group proteins (p &lt; 0.05, FC &gt; 2), and four out of the 14 histone proteins were significantly enriched in emboli from patients with AF (p &lt; 0.05, FC &gt; 2). The most significantly represented functional GO pathways in patients with AF involved PMN activation and degranulation (p &lt; 10-7). CONCLUSIONS: The enrichment of PMN proteins and histones, as well as pathways culminating in PMN activation and degranulation, suggests the presence of neutrophil extracellular traps (NETs) in emboli of stroke patients with AF. NETs are a significant though understudied structural component of clots, and so this work hints at not only significant pathological immunothrombosis in patients with AF, but also at potential therapeutic targets for AIS in this population.

Screening for urinary markers predicting hematopoietic stem cell injury induced by busulfan using genetically diverse mice

Busulfan (BU) is an alkylating agent used as a conditioning agent prior to hematopoietic stem cell (HSC) transplantation as it is known to be cytotoxic to host hematopoietic stem and progenitor cells. The susceptibility of HSCs to BU injury plays an important role in the myeloablative efficacy of BU. Different susceptibilities were demonstrated in genetically diverse (GD) mice in our preliminary research. Three strains of GD mice with different susceptibilities to BU-induced HSC injury were used for screening biological markers of HSC injury susceptibility in urine. The urine proteins were analyzed using liquid chromatography coupled with tandem mass spectrometry to screen for differentially expressed proteins. Screening for possible biomarkers based on differences in protein expression abundance was validated using enzyme-linked immunoassay (ELISA). Functional analysis showed that the differential proteins were all involved in a series of biological pathways related to cellular senescence, apoptosis, and angiogenesis; whereas the differential proteins of the high-susceptible strain were enriched for the regulation of bone marrow microenvironment pathways, those of low-susceptible strain were enriched for the proapoptotic effect of GTPase pathways. Based on protein abundance differences, several urinary proteins that may be indicative of susceptibility were screened, and ELISA validation results showed that angiotensin-converting enzyme may be a potential biomarker predicting HSC susceptibility for BU conditioning. This study indicates that urinary protein levels can reflect differences in susceptibility to BU-induced HSC injury. Using GD mice to construct genetic difference models will provide preclinical data for screening BU-related biological markers.

Effects of GHR Deficiency and Juvenile Hypoglycemia on Immune Cells of a Porcine Model for Laron Syndrome

Laron syndrome (LS) is a rare genetic disorder characterized by low levels of insulin-like growth factor 1 (IGF1) and high levels of growth hormone (GH) due to mutations in the growth hormone receptor gene (GHR). A GHR-knockout (GHR-KO) pig was developed as a model for LS, which displays many of the same features as humans with LS-like transient juvenile hypoglycemia. This study aimed to investigate the effects of impaired GHR signaling on immune functions and immunometabolism in GHR-KO pigs. GHR are located on various cell types of the immune system. Therefore, we investigated lymphocyte subsets, proliferative and respiratory capacity of peripheral blood mononuclear cells (PBMCs), proteome profiles of CD4- and CD4+ lymphocytes and IFN-α serum levels between wild-type (WT) controls and GHR-KO pigs, which revealed significant differences in the relative proportion of the CD4+CD8α- subpopulation and in IFN-α levels. We detected no significant difference in the respiratory capacity and the capacity for polyclonal stimulation in PBMCs between the two groups. But proteome analysis of CD4+ and CD4- lymphocyte populations revealed multiple significant protein abundance differences between GHR-KO and WT pigs, involving pathways related to amino acid metabolism, beta-oxidation of fatty acids, insulin secretion signaling, and oxidative phosphorylation. This study highlights the potential use of GHR-KO pigs as a model for studying the effects of impaired GHR signaling on immune functions.

Proteomic analysis of small intestinal neuroendocrine tumors and mesenteric fibrosis.

Mesenteric metastases in small intestinal neuroendocrine tumors (SI-NETs) are associated with mesenteric fibrosis (MF) in a proportion of patients. MF can induce severe abdominal complications, and an effective preventive treatment is lacking. To elucidate possible novel therapeutic targets, we performed a proteomics-based analysis of MF. The tumor cell and stromal compartment of primary tumors and paired mesenteric metastases of SI-NET patients with MF (n = 6) and without MF (n = 6) was analyzed by liquid chromatography-mass spectrometry-based proteomics. Analysis of differential protein abundance was performed. Collagen alpha-1(XII) (COL12A1) and complement component C9 (C9) expression was evaluated by immunohistochemistry (IHC) in mesenteric metastases. A total of 2988 proteins were identified. Unsupervised hierarchical clustering showed close clustering of paired primary and mesenteric tumor cell samples. Comparing MF to non-MF samples, we detected differentially protein abundance solely in the mesenteric metastasis stroma group. There was no differential abundance of proteins in tumor cell samples or primary tumor stroma samples. Analysis of the differentially abundant proteins (n = 36) revealed higher abundance in MF samples of C9, various collagens and proteoglycans associated with profibrotic extracellular matrix dysregulation and signaling pathways. Proteins involved in fatty acid oxidation showed a lower abundance. COL12A1 and C9 were confirmed by IHC to have significantly higher expression in MF mesenteric metastases compared to non-MF. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development.

NaRnEA: An Information Theoretic Framework for Gene Set Analysis

Gene sets are being increasingly leveraged to make high-level biological inferences from transcriptomic data; however, existing gene set analysis methods rely on overly conservative, heuristic approaches for quantifying the statistical significance of gene set enrichment. We created Nonparametric analytical-Rank-based Enrichment Analysis (NaRnEA) to facilitate accurate and robust gene set analysis with an optimal null model derived using the information theoretic Principle of Maximum Entropy. By measuring the differential activity of ~2500 transcriptional regulatory proteins based on the differential expression of each protein's transcriptional targets between primary tumors and normal tissue samples in three cohorts from The Cancer Genome Atlas (TCGA), we demonstrate that NaRnEA critically improves in two widely used gene set analysis methods: Gene Set Enrichment Analysis (GSEA) and analytical-Rank-based Enrichment Analysis (aREA). We show that the NaRnEA-inferred differential protein activity is significantly correlated with differential protein abundance inferred from independent, phenotype-matched mass spectrometry data in the Clinical Proteomic Tumor Analysis Consortium (CPTAC), confirming the statistical and biological accuracy of our approach. Additionally, our analysis crucially demonstrates that the sample-shuffling empirical null models leveraged by GSEA and aREA for gene set analysis are overly conservative, a shortcoming that is avoided by the newly developed Maximum Entropy analytical null model employed by NaRnEA.

Maternal and Intrauterine Influences on Feto-Placental Growth Are Accompanied by Sexually Dimorphic Changes in Placental Mitochondrial Respiration, and Metabolic Signalling Pathways.

Adverse maternal environments such as small size, malnutrition, and metabolic conditions are known to influence fetal growth outcomes. Similarly, fetal growth and metabolic alterations may alter the intrauterine environment and affect all fetuses in multiple gestation/litter-bearing species. The placenta is the site of convergence between signals derived from the mother and the developing fetus/es. Its functions are fuelled by energy generated by mitochondrial oxidative phosphorylation (OXPHOS). The aim of this study was to delineate the role of an altered maternal and/or fetal/intrauterine environment in feto-placental growth and placental mitochondrial energetic capacity. To address this, in mice, we used disruptions of the gene encoding phosphoinositol 3-kinase (PI3K) p110α, a growth and metabolic regulator to perturb the maternal and/or fetal/intrauterine environment and study the impact on wildtype conceptuses. We found that feto-placental growth was modified by a perturbed maternal and intrauterine environment, and effects were most evident for wildtype males compared to females. However, placental mitochondrial complex I+II OXPHOS and total electron transport system (ETS) capacity were similarly reduced for both fetal sexes, yet reserve capacity was additionally decreased in males in response to the maternal and intrauterine perturbations. These were also sex-dependent differences in the placental abundance of mitochondrial-related proteins (e.g., citrate synthase and ETS complexes), and activity of growth/metabolic signalling pathways (AKT and MAPK) with maternal and intrauterine alterations. Our findings thus identify that the mother and the intrauterine environment provided by littermates modulate feto-placental growth, placental bioenergetics, and metabolic signalling in a manner dependent on fetal sex. This may have relevance for understanding the pathways leading to reduced fetal growth, particularly in the context of suboptimal maternal environments and multiple gestation/litter-bearing species.

Quantitative Proteomics Reveal the Inherent Antibiotic Resistance Mechanism against Norfloxacin Resistance in Aeromonas hydrophila.

Recently, the prevalence of Aeromonas hydrophila antibiotic-resistant strains has been reported in aquaculture, but its intrinsic antibiotic resistance mechanisms are largely unknown. In the present study, a label-free proteomics technology was used to compare the differential protein abundances in response to norfloxacin (NOR) stress in A. hydrophila. The results showed that there were 186 proteins decreasing and 220 proteins increasing abundances in response to NOR stress. Bioinformatics analysis showed that the differentially expressed proteins were enriched in several biological processes, such as sulfur metabolism and homologous recombination. Furthermore, the antibiotic sensitivity assays showed that the deletion of AHA_0904, cirA, and cysI significantly decreased the resistance against NOR, whereas ΔAHA_1239, ΔcysA, ΔcysD, and ΔcysN significantly increased the resistance against NOR. Our results provide insights into NOR resistance mechanisms and indicate that AHA_0904, cirA, AHA_1239, and sulfur metabolism may play important roles in NOR resistance in A. hydrophila.

Proteotranscriptomic Discrimination of Tumor and Normal Tissues in Renal Cell Carcinoma.

Clear cell renal carcinoma is the most frequent type of kidney cancer, with an increasing incidence rate worldwide. In this research, we used a proteotranscriptomic approach to differentiate normal and tumor tissues in clear cell renal cell carcinoma (ccRCC). Using transcriptomic data of patients with malignant and paired normal tissue samples from gene array cohorts, we identified the top genes over-expressed in ccRCC. We collected surgically resected ccRCC specimens to further investigate the transcriptomic results on the proteome level. The differential protein abundance was evaluated using targeted mass spectrometry (MS). We assembled a database of 558 renal tissue samples from NCBI GEO and used these to uncover the top genes with higher expression in ccRCC. For protein level analysis 162 malignant and normal kidney tissue samples were acquired. The most consistently upregulated genes were IGFBP3, PLIN2, PLOD2, PFKP, VEGFA, and CCND1 (p < 10-5 for each gene). Mass spectrometry further validated the differential protein abundance of these genes (IGFBP3, p = 7.53 × 10-18; PLIN2, p = 3.9 × 10-39; PLOD2, p = 6.51 × 10-36; PFKP, p = 1.01 × 10-47; VEGFA, p = 1.40 × 10-22; CCND1, p = 1.04 × 10-24). We also identified those proteins which correlate with overall survival. Finally, a support vector machine-based classification algorithm using the protein-level data was set up. We used transcriptomic and proteomic data to identify a minimal panel of proteins highly specific for clear cell renal carcinoma tissues. The introduced gene panel could be used as a promising tool in the clinical setting.

Study of Melipona quadrifasciata brain under operant learning using proteomic and phosphoproteomic analysis.

Learning to anticipate events based on the predictive relationship between an action and an outcome (operant conditioning) is a form of associative learning shared by humans and most of other living beings, including invertebrates. Several behavioral studies on the mechanisms of operant conditioning have included Melipona quadrifasciata, a honey bee that is easily manipulated due to lack of sting. In this work, brain proteomes of Melipona bees trained using operant conditioning and untrained (control) bees were compared by two-dimensional gel electrophoresis analysis within pI range of 3-10 and 4-7; in order to find proteins specifically related to this type of associative learning.One protein was detected with differential protein abundance in the brains of trained bees, when compared to not trained ones, through computational gel imaging and statistical analysis. This protein was identified by peptide mass fingerprinting and MS/MS peptide fragmentation using a MALDI-TOF/TOF mass spectrometer as one isoform of arginine kinase monomer, apparently dephosphorylated. Brain protein maps were obtained by 2-DE (Two-dimensional gel electrophoresis) from a total proteins and phosphoproteins extract of the bee Melipona quadrifasciata. One isoform of arginine kinase, probably a dephosphorylated isoform, was significantly more abundant in the brain of trained bees using operant conditioning. Arginine kinase has been reported as an important enzyme of the energy releasing process in the visual system of the bee, but it may carry out additional and unexpected functions in the bee brain for learning process.

Quantitative Proteomic Analysis of Serum Reveals MST1 as a Potential Candidate Biomarker in Spontaneously Diabetic Cynomolgus Monkeys.

The prevalence of type 2 diabetes (T2DM) is increasing globally, creating essential demands for T2DM animal models for the study of disease pathogenesis, prevention, and therapy. A non-human primate model such as cynomolgus monkeys can develop T2DM spontaneously in an age-dependent way similar to humans. In this study, a data-independent acquisition-based quantitative proteomics strategy was employed to investigate the serum proteomic profiles of spontaneously diabetic cynomolgus monkeys compared with healthy controls. The results revealed significant differences in protein abundances. A total of 95 differentially expressed proteins (DEPs) were quantitatively identified in the current study, among which 31 and 64 proteins were significantly upregulated and downregulated, respectively. Bioinformatic analysis revealed that carbohydrate digestion and absorption was the top enriched pathway by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Protein-protein interaction network analysis demonstrated that MST1 was identified as the most connected protein in the network and could be considered as the hub protein. MST1 was significantly and inversely associated with FSG and HbA1c. Furthermore, recent lines of evidence also indicate that MST1 acts as a crucial regulator in regulating hepatic gluconeogenesis to maintain metabolic homeostasis while simultaneously suppressing the inflammatory processes. In conclusion, our study provides novel insights into serum proteome changes in spontaneously diabetic cynomolgus monkeys and points out that the dysregulation of several DEPs may play an important role in the pathogenesis of T2DM.

Calcyclin-binding protein-promoted degradation of MdFRUCTOKINASE2 regulates sugar homeostasis in apple.

Fructokinase (FRK) activates fructose through phosphorylation, which sends the activated fructose into primary metabolism and regulates fructose signaling capabilities in plants. The apple (Malus × domestica) FRK gene MdFRK2 shows especially high affinity to fructose, and its overexpression decreases fructose levels in the leaves of young plants. However, in the current study of mature plants, fruits of transgenic apple trees overexpressing MdFRK2 accumulated a higher level of fructose than wild-type (WT) fruits (at both young and mature stages). Transgenic apple trees with high mRNA MdFRK2 expression showed no significant differences in MdFRK2 protein abundance or FRK enzyme activity compared to WT in mature leaves, young fruits, and mature fruits. Immunoprecipitation-mass spectrometry analysis identified an skp1, cullin, F-box (SCF) E3 ubiquitin ligase, calcyclin-binding protein (CacyBP), that interacted with MdFRK2. RNA-sequencing analysis provided evidence for ubiquitin-mediated post-transcriptional regulation of MdFRK2 protein for the maintenance of fructose homeostasis in mature leaves and fruits. Further analyses suggested an MdCacyBP-MdFRK2 regulatory module, in which MdCacyBP interacts with and ubiquitinates MdFRK2 to facilitate its degradation by the 26S proteasome, thus decreasing the FRK enzyme activity to elevate fructose concentration in transgenic apple trees. This result uncovered an important mechanism underlying plant fructose homeostasis in different organs through regulating the MdFRK2 protein level via ubiquitination and degradation. Our study provides usable data for the future improvement of apple flavor and expands our understanding of the molecular mechanisms underlying plant fructose content and signaling regulation.

Two-Dye Versus Three-Dye DIGE for Comparative Testis Tissue Proteomic Analysis.

The global analysis of the proteome is an important tool in cell biology. Comparative proteomic evaluations can identify and compare the composition, dynamics, and modifications between different samples. Comparing tissue proteomes under different conditions is crucial for advancing the biomedical field. Fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) is a sensitive and robust biochemical method that can compare multiple protein samples over a broad dynamic range on the same analytical gel and can be used to establish differentially expressed protein profiles between different sample groups. 2D-DIGE involves fluorescently labeling protein samples with CyDye flours, via a two-dye or a three-dye system, pre-separation by isoelectric point, and molecular weight. DIGE circumvents gel-to-gel variability by multiplexing samples to a single gel and through the use of a pooled internal standard for normalization, thus enabling accurate high-resolution analysis of differences in protein abundance between samples. This chapter discusses 2D-DIGE as a comparative tissue proteomic technique and describes in detail the experimental steps required for comparative proteomic analysis employing both options of two-dye and three-dye DIGE minimal labeling.