Quantitative Proteomics Approach Research Articles

Identification and Validation of Yak (Bos grunniens) Frozen-Thawed Sperm Proteins Associated with Capacitation and the Acrosome Reaction.

To achieve fertilization, mammalian spermatozoa must undergo capacitation and the acrosome reaction (AR) within the female reproductive tract. However, the effects of cryopreservation on sperm maturation and fertilizing potential have yet to be established. To gain insight into changes in protein levels within sperm cells prepared for use in the context of fertilization, a comprehensive quantitative proteomic profiling approach was used to analyze frozen-thawed Ashidan yak spermatozoa under three sequential conditions: density gradient centrifugation-based purification, incubation in a capacitation medium, and treatment with the calcium ionophore A23187 to facilitate AR induction. In total, 3280 proteins were detected in these yak sperm samples, of which 3074 were quantified, with 68 and 32 being significantly altered following sperm capacitation and AR induction. Differentially abundant capacitation-related proteins were enriched in the metabolism and PPAR signaling pathways, while differentially abundant AR-related proteins were enriched in the AMPK signaling pathway. These data confirmed a role for superoxide dismutase 1 (SOD1) as a regulator of sperm capacitation while also offering indirect evidence that heat shock protein 90 alpha (HSP90AA1) regulates the AR. Together, these findings offer a means whereby sperm fertility-related marker proteins can be effectively identified. Data are available via Proteome Xchange with identifier PXD035038.

6-Methylcoumarin rescues bacterial quorum sensing induced ribosome-inactivating stress in Caenorhabditis elegans

IntroductionBacterial pathogenicity has for long posed severe effects on patient care. Pseudomonas aeruginosa is a common cause of hospital-acquired infections and nosocomial illnesses. It is known to infect the host by colonizing through quorum sensing and the production of exotoxins. MethodsThe current effort is an analysis of proteomic alterations caused by P. aeruginosa PAO1 to study the effects of quorum sensing inhibitor 6-Methylcoumarin on PAO1 infectivity in the Caenorhabditis elegans model. ResultsThrough tandem mass tag-based quantitative proteomics approaches, 229 proteins were found to be differentially regulated in infection and upon inhibition. Among these, 34 proteins were found to be dysregulated in both infection and quorum-sensing inhibition conditions. Along with the dysregulation of proteins involved in host-pathogen interaction, PAO1 was found to induce ribosome-inactivating stress accompanied by the downregulating mitochondrial proteins. This in turn caused dysregulation of apoptosis. The expression of multiple proteins involved in ribosome biogenesis and structure, oxidative phosphorylation, and mitochondrial enzymes were altered due to infection. This mechanism, adapted by PAO1 to survive in the host, was inhibited by 6-Methylcoumarin by rescuing the downregulation of ribosomal and mitochondrial proteins. ConclusionsTaken together, the data reflect the molecular alterations due to quorum sensing and the usefulness of inhibitors in controlling pathogenesis.

TMT-based quantitative proteomic analysis of indica rice cultivars reveals that novel components of the signaling pathways might play a role in grain length regulation

Grain length is one of the most important rice grain appearance components. To better understand the protein regulated by grain length in indica rice, the tandem mass tag (TMT) labeling combined with LC-MS/MS analysis was used for quantitative identification of differentially regulated proteins by comparing six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB) to the short-grain cultivar BoB, respectively. A total of 6622 proteins were detected for quantitative analysis by comparing protein content of six long-grain cultivars to the short-grain cultivar, and 715 proteins were significantly regulated, consisting of 336 uniquely over-accumulated proteins and 355 uniquely down-accumulated proteins. KEGG pathway analysis revealed that most of accumulated proteins are involved in metabolic pathways, biosynthesis of secondary metabolites and phenylpropanoid biosynthesis. Four down-accumulated proteins maybe involved in the signaling pathways for grain length regulation. LC-PRM/MS quantitative analysis was used to analyze 10 differentially expressed proteins. The results were almost consistent with the TMT quantitative analysis. qRT-PCR analysis results showed that the transcription level was not always parallel to the protein content. This study identified many novel grain length accumulated proteins through the quantitative proteomics approach, providing candidate genes for further study of grain size regulatory mechanisms. SignificanceRice grain length is one of the most important characteristics influencing appearance and yield. Six long-grain cultivars (MeiB, LongfengB, YexiangB, FengtianB, WantaiB, and DingxiangB obtained in Guangxi province of China from the 2000s to 2020s) and one short-grain cultivar (BoB obtained in Guangxi province of China in 1980s) were used for comparative analyses. Totally, 715 differentially expressed proteins (DEPs) were identified using TMT-base proteomic analysis. The numbers of DEPs increased as the grain length increased. 4 DEPs may be related to rice's signaling pathways for grain size regulation. A total of 85 DEPs regulated in at least four long-grain cultivars compared with the short-grain cultivar BoB, and 7 proteins were over-accumulated, and 3 proteins were down-accumulated in six long-grain cultivars. These findings provide valuable information to better understand the mechanisms of protein regulation by grain length in rice.

Serum and urine profiling by high-throughput TMT-based proteomics for the investigation of renal dysfunction in canine babesiosis

Canine babesiosis is a tick-borne disease caused by Babesia canis, with acute kidney injury as one of the common complications. In the study 8 healthy control dogs and 22 dogs with naturally occurring babesiosis were enrolled, with the aim to analyse differences in serum and urinary proteomes between healthy dogs and dogs with different degree of renal dysfunction in babesiosis using a label-based high-throughput quantitative proteomic approach. In serum, 58 proteins were found differentially abundant between healthy controls and groups of dogs with different degrees of renal dysfunction in babesiosis, while in urine there were 259 differentially abundant proteins. In addition, altered biological pathways were detected in the diseased dogs using bioinformatics tools and validation of several candidate biomarkers was performed. SignificanceThe main aim of this comprehensive study was to perform analyses of serum and urinary proteomes of dogs with renal dysfunction in babesiosis compared to healthy dogs using, for the first time, a high-throughput proteomic method and functional enrichment analyses. Serum and urine samples of the same dogs were investigated in order to gain a more complete picture of pathologic changes taking place in renal dysfunction in babesiosis. We highlighted two putative biomarkers validated herein which could be of importance for early diagnosis of renal dysfunction in canine babesiosis, as they are easily accessible from urine and their concentration rises before the appearance of azotaemia: urinary neutrophil gelatinase-associated lipocalin (NGAL) and urinary liver-type fatty acid-binding protein (L-FABP).

Exploration of the underlying mechanisms of isoniazid/rifampicin-induced liver injury in mice using an integrated proteomics and metabolomics approach.

The hepatotoxic mechanism resulting from coadministration of isoniazid (INH) and rifampicin (RIF) are complex and studies remain inconclusive. To systematically explore the underlying mechanisms, an integrated mass-based untargeted metabolomics and label-free quantitative proteomics approach was used to clarify the mechanism of INH/RIF-induced liver injury. Thirty male mice were randomly divided into three groups: control (receiving orally administered vehicle solution), INH (150 mg/kg) + RIF (300 mg/kg) orally administered for either 7or 14 days, respectively. Serum was collected for the analysis of biochemical parameters and liver samples were obtained for massspectrum-based proteomics, metabolomics, and lipidomics analysis. Overall, 511 proteins, 31 metabolites, and 23 lipids were dysregulated and identified, and disordered biological pathways were identified. The network of integrated multiomics showed that glucose, lipid, and amino acid metabolism as well as energy metabolism were mainly dysregulated and led to oxidative stress, inflammation, liver steatosis, and cell death induced by INH and RIF. Coadministration of INH and RIF can induce liver injury by oxidative stress, inflammation, liver steatosis, and cell death, and the reduction in glutathione levels may play a critical role in these systematic changes and warrants further study.

Identification of perioperative neurocognitive dysfunction biomarkers in cerebrospinal fluid with quantitative proteomic approach in patients undergoing transurethral resection of prostate with combined spinal and epidural analgesia.

This study aimed to identify predictive biomarkers of perioperative neurocognitive dysfunction (PND) in cerebrospinal fluid of elderly male patients undergoing elective transurethral resection of prostate, using an isobaric tags for relative and absolute quantitative-based quantitative proteomic approach. Patients were evaluated with Mini Mental State Examination at -1 and+3 days of operation. Presence of PND was determined with Z-score method. Patients characteristics and quantitative cerebrospinal fluid proteomes detected with isobaric tags for relative and absolute quantitative-were compared between PND and non-PND patients. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis were performed to identify pathways potentially involved in PND. A total of 229 patients were included in the study and 32 were diagnosed with PND (incidence 14.4%). The age, incidence of hypertension, and diabetes of PND patients were significantly higher than non-PND patients (P < .05). There were 85 differentially expressed proteins identified, among which High Mobility Group Box 1, prostaglandin D synthase, and matrix metalloproteinase inhibitor were considered to be promising candidates as they might play important roles in pathophysiology of PND. Proteomic approach identified potential biomarkers for predicting the occurrence of PND. These findings need to be validated in further studies.

ALK fusion promotes metabolic reprogramming of cancer cells by transcriptionally upregulating PFKFB3.

Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase of the insulin receptor kinase subfamily, is activated in multiple cancer types through translocation or overexpression. Although several generations of ALK tyrosine kinase inhibitors (TKIs) have been developed for clinic use, drug resistance remains a major challenge. In this study, by quantitative proteomic approach, we identified the glycolytic regulatory enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), as a new target of ALK. Expression of PFKFB3 is highly dependent on ALK activity in ALK+ anaplastic large cell lymphoma and non-small-cell lung cancer (NSCLC) cells. Notably, ALK and PFKFB3 expressions exhibit significant correlation in clinic ALK+ NSCLC samples. We further demonstrated that ALK promotes PFKFB3 transcription through the downstream transcription factor STAT3. Upregulation of PFKFB3 by ALK is important for high glycolysis level as well as oncogenic activity of ALK+ lymphoma cells. Finally, targeting PFKFB3 by its inhibitor can overcome drug resistance in cells bearing TKI-resistant mutants of ALK. Collectively, our studies reveal a novel ALK-STAT3-PFKFB3 axis to promote cell proliferation and tumorigenesis, providing an alternative strategy for the treatment of ALK-positive tumors.

Quantitative proteomic analytic approaches to identify metabolic changes in the medial prefrontal cortex of rats exposed to space radiation.

NASA’s planned mission to Mars will result in astronauts being exposed to ∼350 mSv/yr of Galactic Cosmic Radiation (GCR). A growing body of data from ground-based experiments indicates that exposure to space radiation doses (approximating those that astronauts will be exposed to on a mission to Mars) impairs a variety of cognitive processes, including cognitive flexibility tasks. Some studies report that 33% of individuals may experience severe cognitive impairment. Translating the results from ground-based rodent studies into tangible risk estimates for astronauts is an enormous challenge, but it would be germane for NASA to use the vast body of data from the rodent studies to start developing appropriate countermeasures, in the expectation that some level of space radiation (SR) -induced cognitive impairment could occur in astronauts. While some targeted studies have reported radiation-induced changes in the neurotransmission properties and/or increased neuroinflammation within space radiation exposed brains, there remains little information that can be used to start the development of a mechanism-based countermeasure strategy. In this study, we have employed a robust label-free mass spectrometry (MS) -based untargeted quantitative proteomic profiling approach to characterize the composition of the medial prefrontal cortex (mPFC) proteome in rats that have been exposed to 15 cGy of 600 MeV/n28Si ions. A variety of analytical techniques were used to mine the generated expression data, which in such studies is typically hampered by low and variable sample size. We have identified several pathways and proteins whose expression alters as a result of space radiation exposure, including decreased mitochondrial function, and a further subset of proteins differs in rats that have a high level of cognitive performance after SR exposure in comparison with those that have low performance levels. While this study has provided further insight into how SR impacts upon neurophysiology, and what adaptive responses can be invoked to prevent the emergence of SR-induced cognitive impairment, the main objective of this paper is to outline strategies that can be used by others to analyze sub-optimal data sets and to identify new information.

Proteomic changes in various organs of Haemaphysalis longicornis under long-term starvation.

Haemaphysalis longicornis (Neumann), a tick of public health and veterinary importance, spend the major part of their life cycle off-host, especially the adult host-seeking period. Thus, they have to contend with prolonged starvation. Here, we investigated the underlying molecular mechanism of tick starvation endurance in the salivary glands, midguts, ovaries, and Malpighian tubules of starved H. longicornis ticks using the data-independent acquisition quantitative proteomic approach to study the proteome changes. Essential synthases such as glutamate synthase, citrate synthase, and ATP synthase were up-regulated probably due to increased proteolysis and amino acid catabolism during starvation. The up-regulation of succinate dehydrogenase, ATP synthase, cytochrome c oxidase, and ADP/ATP translocase closely fits with an increased oxidative phosphorylation function during starvation. The differential expression of superoxide dismutase, glutathione reductase, glutathione S-transferase, thioredoxin, and peroxiredoxin indicated fasting-induced oxidative stress. The up-regulation of heat shock proteins could imply the activation of a protective mechanism that checks excessive protein breakdown during starvation stress. The results of this study could provide useful information about the vulnerabilities of ticks that could aid in tick control efforts.

Biomarkers in Neurodegenerative Diseases: Proteomics Spotlight on ALS and Parkinson's Disease.

Neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are both characterized by pathogenic protein aggregates that correlate with the progressive degeneration of neurons and the loss of behavioral functions. Both diseases lack biomarkers for diagnosis and treatment efficacy. Proteomics is an unbiased quantitative tool capable of the high throughput quantitation of thousands of proteins from minimal sample volumes. We review recent proteomic studies in human tissues, plasma, cerebrospinal fluid (CSF), and exosomes in ALS and PD that identify proteins with potential utility as biomarkers. Further, we review disease-related post-translational modifications in key proteins TDP43 in ALS and α-synuclein in PD studies, which may serve as biomarkers. We compare relative and absolute quantitative proteomic approaches in key biomarker studies in ALS and PD and discuss recent technological advancements which may identify suitable biomarkers for the early-diagnosis treatment efficacy of these diseases.

Polyphenols from Thinned Young Apples: HPLC-HRMS Profile and Evaluation of Their Anti-Oxidant and Anti-Inflammatory Activities by Proteomic Studies.

The qualitative profile of thinned apple polyphenols (TAP) fraction (≈24% of polyphenols) obtained by purification through absorbent resin was fully investigated by LC-HRMS in positive and negative ion mode and using ESI source. A total of 68 polyphenols were identified belonging to six different classes: flavanols, flavonols, dihydrochalchones, flavanones, flavones and organic and phenolic acids. The antioxidant and anti-inflammatory activities were then investigated in cell models with gene reporter for NRF2 and NF-κB and by quantitative proteomic (label-free and SILAC) approaches. TAP dose-dependently activated NRF2 and in the same concentration range (10–250 µg/mL) inhibited NF-κB nuclear translocation induced by TNF-α and IL-1α as pro-inflammatory promoters. Proteomic studies elucidated the molecular pathways evoked by TAP treatment: activation of the NRF2 signaling pathway, which in turn up-regulates protective oxidoreductases and their nucleophilic substrates such as GSH and NADPH, the latter resulting from the up-regulation of the pentose phosphate pathway. The increase in the enzymatic antioxidant cellular activity together with the up-regulation of the heme-oxygenase would explain the anti-inflammatory effect of TAP. The results suggest that thinned apples can be considered as a valuable source of apple polyphenols to be used in health care products to prevent/treat oxidative and inflammatory chronic conditions.

Protein Profiling of a Cellular Model of NAFLD by Advanced Bioanalytical Approaches.

Advanced quantitative bioanalytical approaches in combination with network analyses allow us to answer complex biological questions, such as the description of changes in protein profiles under disease conditions or upon treatment with drugs. In the present work, three quantitative proteomic approaches—either based on labelling or not—in combination with network analyses were applied to a new in vitro cellular model of nonalcoholic fatty liver disease (NAFLD) for the first time. This disease is characterized by the accumulation of lipids, inflammation, fibrosis, and insulin resistance. Hepatic G2 cells were used as model, and NAFLD was induced by a complex of oleic acid and bovine albumin. The development of the disease was verified by lipid vesicle staining and by the increase in the expression of perilipin-2—a protein constitutively present in the vesicles during NAFLD. The nLC–MS/MS analyses of peptide samples obtained from three different proteomic approaches resulted in accurate and reproducible quantitative data of protein fold-change expressed in NAFLD versus control cells. The differentially regulated proteins were used to evaluate the involved and statistically enriched pathways. Network analyses highlighted several functional and disease modules affected by NAFLD, such as inflammation, oxidative stress defense, cell proliferation, and ferroptosis. Each quantitative approach allowed the identification of similar modulated pathways. The combination of the three approaches improved the power of statistical network analyses by increasing the number of involved proteins and their fold-change. In conclusion, the application of advanced bioanalytical approaches in combination with pathway analyses allows the in-depth and accurate description of the protein profile of an in vitro cellular model of NAFLD by using high-resolution quantitative mass spectrometry data. This model could be extremely useful in the discovery of new drugs to modulate the equilibrium NAFLD health state.

IFITM3 promotes malignant progression, cancer stemness and chemoresistance of gastric cancer by targeting MET/AKT/FOXO3/c-MYC axis

BackgroundTargeting the HGF/MET signaling pathway has been a viable therapeutic strategy for various cancer types due to hyperactivation of HGF/MET axis occurs frequently that leads to detrimental cancer progression and recurrence. Deciphering novel molecule mechanisms underlying complex HGF/MET signaling network is therefore critical to development of effective therapeutics for treating MET-dependent malignancies.ResultsUsing isobaric mass tag-based quantitative proteomics approach, we identified IFITM3, an interferon-induced transmembrane protein that was highly expressed in micro-dissected gastric cancer (GC) tumor regions relative to adjacent non-tumor epithelia. Analyses of GC clinical specimens revealed that expression IFITM3 was closely correlated to advanced pathological stages. IFITM3 has been reported as a PIP3 scaffold protein that promotes PI3K signaling. In present study, we unprecedentedly unraveled that IFITM3 associated with MET and AKT to facilitate HGF/MET mediated AKT signaling crosstalk in suppressing FOXO3, consequently leading to c-MYC mediated GC progression. In addition, gene ontology analyses of the clinical GC cohort revealed significant correlation between IFITM3-associated genes and targets of c-MYC, which is a crucial downstream effector of HGF/MET pathway in cancer progression. Moreover, we demonstrated ectopic expression of IFITM3 suppressed FOXO3 expression, consequently led to c-MYC induction to promote tumor growth, cell metastasis, cancer stemness as well as chemoresistance. Conversely, depletion of IFITM3 resulted in suppression of HGF triggered cellular growth and migration via inhibition of AKT/c-MYC signaling in GC.ConclusionsIn summary, our present study unveiled a novel regulatory mechanism for c-MYC-driven oncogenesis underlined by IFITM3-mediated signaling crosstalk between MET associated AKT signaling cascade.

Proteomic Discovery and Validation of Novel Fluid Biomarkers for Improved Patient Selection and Prediction of Clinical Outcomes in Alzheimer's Disease Patient Cohorts.

Alzheimer’s disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive decline. The two cardinal neuropathological hallmarks of AD include the buildup of cerebral β amyloid (Aβ) plaques and neurofibrillary tangles of hyperphosphorylated tau. The current disease-modifying treatments are still not effective enough to lower the rate of cognitive decline. There is an urgent need to identify early detection and disease progression biomarkers that can facilitate AD drug development. The current established readouts based on the expression levels of amyloid beta, tau, and phospho-tau have shown many discrepancies in patient samples when linked to disease progression. There is an urgent need to identify diagnostic and disease progression biomarkers from blood, cerebrospinal fluid (CSF), or other biofluids that can facilitate the early detection of the disease and provide pharmacodynamic readouts for new drugs being tested in clinical trials. Advances in proteomic approaches using state-of-the-art mass spectrometry are now being increasingly applied to study AD disease mechanisms and identify drug targets and novel disease biomarkers. In this report, we describe the application of quantitative proteomic approaches for understanding AD pathophysiology, summarize the current knowledge gained from proteomic investigations of AD, and discuss the development and validation of new predictive and diagnostic disease biomarkers.

Easier, More Robust, and Faster Low-flow Quantitative Proteomics Approach

Easier, More Robust, and Faster Low-flow Quantitative Proteomics Approach

Identification of novel biomarkers and therapeutic target candidates for stasis-heat symptom pattern of acute intracerebral hemorrhage by quantitative plasma proteomics.

To explore the novel biomarkers and therapeutic target candidates related to the stasis-heat syndrome of acute intracerebral hemorrhage (AICH). Applying an isobaric tagging for relative and absolute quantitation-(iTRAQ-) based quantitative proteomic approach, plasma samples from AICH patients with stasis-heat, and AICH patients with non-stasis-heat and healthy control subjects were collected and analyzed to distinguish differentially expressed proteins (DEPs) correlated to AICH with stasis-heat in this block design. The standard Western blot was applied to verify DEPs. Additionally, DEPs were analyzed via bioinformatic platforms and further approved via Ingenuity Pathway Analysis (IPA). A total of 26 DEPs were found among AICH with the stasis-heat, AICH with non-stasis-heat, and healthy control group. The seven DEPs compared with the non-stasis-heat group are closely related to the pathogenesis of stasis heat. These proteins showed three different protein expression patterns. The alpha-1-b glycoprotein (A1BG) and copper-protein (CP) were up-regulated in the stasis-heat group, but down-regulated in the non-stasis-heat group. Compared with the non-stasis-heat group, the expression abundance of actinin, alpha 1 (ACTN1), carbonic anhydrase I (CA1), peroxiredoxin 2 (PRDX2), and vinculin (VCL) is higher in the stasis-heat group, while the CD44 is the opposite. These differences reflect that stasis-heat syndrome has more severe inflammatory immune response, coagulation disorders and damage. Bioinformatics analysis revealed that a wide variety of cellular and metabolic processes and some signaling pathways were involved in the pathophysiology of AICH with stasis-heat. AICH with stasis-heat syndrome showed more severe inflammatory reactions, tissue damage, and coagulation disorders than non-stasis heat syndrome. There are differences in the protein expression patterns between the stasis-heat syndrome and non-stasis-heat syndrome. These differences reflect that stasis-heat syndrome has more severe damage. CD44, CP, ACTN1, CA1, VCL, PRDX2, and A1BG could be the potential biomarkers and therapeutic target candidates of the stasis-heat subtype. This study provides a reasonable explaination for Liangxue Tongyu decoction through anti-inflammatory and brain protection treatment.

Differential Proteomic Analysis of Listeria monocytogenes during High-Pressure Processing.

Simple SummaryHigh-pressure processing (HPP) is a prevailing non-thermal food preservation technology. The inactivation mechanisms of Listeria monocytogenes under sub-lethal to lethal damage by different levels of HPP treatments were conducted by label-free quantitative proteomic analysis. HPP might promote translation initiation due to upregulation of most ribosomal subunits and initiation factors. However, protein synthesis was arrested according to the shortage of proteins responsible for elongation, termination and recycling. The quantitative proteomics approaches provide fundamental information on L. monocytogenes under different HPP pressures, and provide theoretical support for HPP against Listeriosis illness and for promotion of safer ready-to-eat foods.High-pressure processing (HPP) is a prevailing non-thermal food preservation technology. The inactivation mechanisms of Listeria monocytogenes under HPP at 200 and 400 MPa for 3 min were investigated by label-free quantitative proteomic analysis and functional enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes. HPP treatment at 400 MPa exhibited significant effects on proteins involved in translation, carbon, carbohydrate, lipid and energy metabolism, and peptidoglycan biosynthesis. HPP increased most ribosomal subunits and initiation factors, suggesting it might shift ribosomal biogenesis to translation initiation. However, protein synthesis was impaired by the shortage of proteins responsible for elongation, termination and recycling. HPP stimulated several ATP-dependent Clp proteases, and the global transcriptional regulator Spx, associating with activation of the stress-activated sigma factor Sigma B (σB) and the transcriptional activator positive regulatory factor A (PrfA) regulons. The quantitative proteomics approaches provide fundamental information on L. monocytogenes under different HPP pressures, and provide theoretical support for HPP against Listeriosis illness and for promotion of safer ready-to-eat foods.

Label-Free Mass Spectrometry-Based Quantitative Proteomics to Evaluate the Effects of the Calcium-Sensing Receptor Agonist Cinacalcet on Protein Expression in Rat Brains and Livers.

BackgroundCinacalcet is a calcium-sensing receptor agonist that is clinically approved for the treatment of secondary hyperparathyroidism in chronic kidney disease and hypercalcemia in patients with parathyroid carcinoma. This study aimed to use quantitative mass spectrometry-based label-free proteomics to evaluate the effects of cinacalcet on protein expression in rat brains and livers.Material/MethodsWe randomly assigned 18 Wistar rats to 2 groups: an untreated control group (n=6) and a group treated with cinacalcet at a dose corresponding to the maximum dose used in humans (2 mg/kg/body weight, 5 days/week) divided into 7-day (n=6) and 21-day (n=6) treatment subgroups. A mass-spectrometry-based label-free quantitative proteomics approach using peptides peak area calculation was used to evaluate the changes in protein expression in examined tissues. Bioinformatics analysis of quantitative proteomics data was done using MaxQuant and Perseus environment.ResultsNo changes in protein expression were revealed in the 7-day treatment subgroup. We detected 10 upregulated and 3 downregulated proteins in the liver and 1 upregulated protein in the brain in the 21-day treatment subgroup compared to the control group. Based on Gene Ontology classification, all identified differentially expressed proteins were indicated as molecular functions involved in the enzyme regulator activity (36%), binding (31%), and catalytic activity (19%).ConclusionsThese findings indicate that long-term cinacalcet therapy can impair phase II of enzymatic detoxication and can cause disturbances in blood hemostasis, lipid metabolism, and inflammatory mediators or contribute to the acceleration of cognitive dysfunction; therefore, appropriate patient monitoring should be considered.

Using a Relative Quantitative Proteomic Method to Identify Differentially Abundant Proteins in Brucella melitensis Biovar 3 and Brucella melitensis M5-90.

Brucellosis, caused by Brucella spp., is one of the most widespread bacterial zoonoses worldwide. Vaccination is still considered the best way to control brucellosis. An investigation into the differential proteome expression patterns of wild and vaccine strains may help researchers and clinicians differentiate between the strains to diagnose and better understand the mechanism(s) underlying differences in virulence. In the present study, a mass spectrometry-based, label-free relative quantitative proteomics approach was used to investigate the proteins expressed by the wild strain, B. melitensis biovar 3 and compare it with those expressed by B. melitensis M5-90. The higher level of virulence for B. melitensis biovar 3 compared to B. melitensis M5-90 was validated in vitro and in vivo. A total of 2133 proteins, encompassing 68% of the theoretical proteome, were identified and quantified by proteomic analysis, resulting in broad coverage of the B. melitensis proteome. A total of 147 proteins were identified as differentially expressed (DE) between these two strains. In addition, 9 proteins and 30 proteins were identified as unique to B. melitensis M5-90 and B. melitensis biovar 3, respectively. Pathway analysis revealed that the majority of the DE proteins were involved in iron uptake, quorum sensing, pyrimidine metabolism, glycine betaine biosynthetic and metabolic processes, thiamine-containing compound metabolism and ABC transporters. The expression of BtpA and VjbR proteins (two well-known virulence factors) in B. melitensis biovar 3 was 8-fold and 2-fold higher than in B. melitensis M5-90. In summary, our results identified many unique proteins that could be selected as candidate markers for differentiating vaccinated animals from animals with wild-type infections. BtpA and VjbR proteins might be responsible for the residual virulence of B. melitensis M5-90, while ABC transporters and thiamine metabolism associated proteins may be newly identified Brucella virulence factors. All of the identified DE proteins provide valuable information for the development of vaccines and the discovery of novel therapeutic targets.

Deciphering the Proteomic Landscape of Mycobacterium tuberculosis in Response to Acid and Oxidative Stresses

The fundamental to the pathogenicity of Mycobacterium tuberculosis (Mtb) is the modulation in the control mechanisms that play a role in sensing and counteracting the microbicidal milieu encompassing various cellular stresses inside the human host. To understand such changes, we measured the cellular proteome of Mtb subjected to different stresses using a quantitative proteomics approach. We identified defined sets of Mtb proteins that are modulated in response to acid and a sublethal dose of diamide and H2O2 treatments. Notably, proteins involved in metabolic, catalytic, and binding functions are primarily affected under these stresses. Moreover, our analysis led to the observations that during acidic stress Mtb enters into energy-saving mode simultaneously modulating the acid tolerance system, whereas under diamide and H2O2 stresses, there were prominent changes in the biosynthesis and homeostasis pathways, primarily modifying the resistance mechanism in diamide-treated bacteria while causing metabolic arrest in H2O2-treated bacilli. Overall, we delineated the adaptive mechanisms that Mtb may utilize under physiological stresses and possible overlap between the responses to these stress conditions. In addition to offering important protein signatures that can be exploited for future mechanistic studies, our study highlights the importance of proteomics in understanding complex adjustments made by the human pathogen during infection.