Label-free Liquid Chromatography-mass Spectrometry Research Articles

Protein-Corona-by-Design in 2D: A Reliable Platform to Decode Bio-Nano Interactions for the Next-Generation Quality-by-Design Nanomedicines.

Hard corona (HC) protein, i.e., the environmental proteins of the biological medium that are bound to a nanosurface, is known to affect the biological fate of a nanomedicine. Due to the size, curvature, and specific surface area (SSA) 3-factor interactions inherited in the traditional 3D nanoparticle, HC-dependent bio–nano interactions are often poorly probed and interpreted. Here, the first HC-by-design case study in 2D is demonstrated that sequentially and linearly changes the HC quantity using functionalized graphene oxide (GO) nanosheets. The HC quantity and HC quality are analyzed using NanoDrop and label-free liquid chromatography–mass spectrometry (LC-MS) followed by principal component analysis (PCA). Cellular responses (uptake and cytotoxicity in J774 cell model) are compared using imaging cytometry and the modified lactate dehydrogenase assays, respectively. Cellular uptake linearly and solely correlates with HC quantity (R2 = 0.99634). The nanotoxicity, analyzed by retrospective design of experiment (DoE), is found to be dependent on the nanomaterial uptake (primary), HC composition (secondary), and nanomaterial exposure dose (tertiary). This unique 2D design eliminates the size–curvature–SSA multifactor interactions and can serve as a reliable screening platform to uncover HC-dependent bio–nano interactions to enable the next-generation quality-by-design (QbD) nanomedicines for better clinical translation.

Functional analysis of synovial fluid from osteoarthritic knee and carpometacarpal joints unravels different molecular profiles.

In this work, we aimed to elucidate the molecular mechanisms driving primary OA. By studying the dynamics of protein expression in two different types of OA joints we searched for similarities and disparities to identify key molecular mechanisms driving OA. For this purpose, human SF samples were obtained from CMC-I OA and knee joint of OA patients. SF samples were analysed by label-free quantitative liquid chromatography mass spectrometry. Disease-relevant proteins identified in proteomics studies, such as clusterin, paraoxonase/arylesterase 1 (PON1) and transthyretin were validated by enzyme-linked immunosorbent assays, and on the mRNA level by droplet digital PCR. Functional studies were performed in vitro using primary chondrocytes. Differential proteomic changes were observed in the concentration of 40 proteins including clusterin, PON1 and transthyretin. Immunoassay analyses of clusterin, PON1, transthyretin and other inflammatory cytokines confirmed significant differences in protein concentration in SF of CMC-I and knee OA patients, with primarily lower protein expression levels in CMC-I. Functional studies on chondrocytes unequivocally demonstrated that stimulation with SF obtained from knee OA, in contrast to CMC-I OA joint, caused a significant upregulation in pro-inflammatory response, cell death and hypertrophy. This study demonstrates that differential expression of molecular players in SF from different OA joints evokes diverse effects on primary chondrocytes. The pathomolecular mechanisms of OA may significantly differ in various joints, a finding that brings a new dimension into the pathogenesis of primary OA.

Peptidome analysis of lung tissues from a hyperoxia-induced bronchopulmonary dysplasia mouse model: Insights into the pathophysiological process of bronchopulmonary dysplasia.

The aim of this study was to identify and compare the peptidomic profiles of lung tissues from neonatal mice with and without bronchopulmonary dysplasia (BPD). Hyperoxia was used to establish the BPD mouse model. Lung tissues obtained on postnatal day (PND) 9 were processed for analysis via histological staining and label-free liquid chromatography-mass spectrometry (LC-MS/MS). Histological analysis of the lung sections from the BPD group showed significant alveolar simplification and aberrant pulmonary vascularization. We identified 3,704 total peptides, of which 63 were differentially expressed in the lung tissues from the BPD group compared with those from the control group. Within this subset, 31 peptides were downregulated, and 32 peptides were upregulated. Bioinformatics analysis suggested several potential roles of the differentially expressed peptides in the pathophysiological process of BPD. In summary, this study highlights novel peptide candidates, and provides new insights for further understanding the molecular mechanism of BPD development.

Proteomic expression profile of injured rat peripheral nerves revealed biological networks and processes associated with nerve regeneration.

Peripheral nerve regeneration is regulated through the coordinated spatio-temporal activation of multiple cellular pathways. In this work, an integrated proteomics and bioinformatics approach was employed to identify differentially expressed proteins at the injury-site of rat sciatic nerve at 20 days after damage. By a label-free liquid chromatography mass-spectrometry (LC-MS/MS) approach, we identified 201 differentially proteins that were assigned to specific canonical and disease and function pathways. These include proteins involved in cytoskeleton signaling and remodeling, acute phase response, and cellular metabolism. Metabolic proteins were significantly modulated after nerve injury to support a specific metabolic demand. In particular, we identified a group of proteins involved in lipid uptake and lipid storage metabolism. Immunofluorescent staining for acyl-CoA diacylglycerol acyltransferase 1 (DGAT1) and DAGT2 expression provided evidence for the expression and localization of these two isoforms in Schwann cells at the injury site in the sciatic nerve. This further supports a specific local regulation of lipid metabolism in peripheral nerve after damage.

Differential Metabolism of a Two-Carbon Substrate by Members of the Paracoccidioides Genus.

The genus Paracoccidioides comprises known fungal pathogens of humans and can be isolated from different infection sites. Metabolic peculiarities in different members of the Paracoccidioides led us to perform proteomic studies in the presence of the two-carbon molecule acetate, which predominates in the nutrient-poor environment of the phagosome. To investigate the expression rates of proteins of different members of Paracoccidioides, including one isolate of P. lutzii (Pb01) and three isolates of P. brasiliensis (Pb03, Pb339, and PbEPM83), using sodium acetate as a carbon source, proteins were quantified using label-free and data-independent liquid chromatography-mass spectrometry. Protein profiles of the isolates were statistically analyzed, revealing proteins that were differentially expressed when the fungus was cultivated in a non-preferential carbon source rather than glucose. A total of 1,160, 1,211, 1,280, and 1,462 proteins were reproducibly identified and relatively quantified in P. lutzii and the P. brasiliensis isolates Pb03, Pb339, and PbEPM83, respectively. Notably, 526, 435, 744, and 747 proteins were differentially expressed among P. lutzii and the P. brasiliensis isolates Pb03, Pb339, and PbEPM83, respectively, with a fold-change equal to or higher than 1.5. This analysis revealed that reorganization of metabolism occurred through the induction of proteins related to gluconeogenesis, glyoxylic/glyoxylate cycle, response to stress, and degradation of amino acids in the four isolates. The following differences were observed among the isolates: higher increases in the expression levels of proteins belonging to the TCA and respiratory chain in PbEPM83 and Pb01; increase in ethanol production in Pb01; utilization of cell wall components for gluconeogenesis in Pb03 and PbEPM83; and increased β-oxidation and methylcitrate cycle proteins in Pb01and PbEPM83. Proteomic profiles indicated that the four isolates reorganized their metabolism in different manners to use acetate as a carbon source.

A Multiplatform Approach for the Discovery of Novel Drug-Induced Kidney Injury Biomarkers.

Drug-induced kidney injury (DIKI) is a common toxicity observed in pharmaceutical development. We demonstrated the use of label-free liquid chromatography-mass spectrometry (LC-MS) and multiplex liquid chromatography-single reaction monitoring (LC-SRM) as practical extensions of standard immunoassay based safety biomarker assessments for identification of new toxicity marker candidates and for improved mechanistic understanding. Two different anticancer drugs, doxorubicin (DOX) and cisplatin (cis-diamminedichloridoplatinum, CDDP), were chosen as the toxicants due to their different modes of nephrotoxicity. Analyses of urine samples from toxicant treated and untreated rats were compared to identify biochemical analytes that changed in response to toxicant exposure. A discovery (label-free LC-MS) and targeted proteomics (multiplex LC-SRM) approach was used in combination with well established immunoassay experiments for the identification of a panel of urinary protein markers related to drug induced nephrotoxicity in rats. The initial generation of an expanded set of markers was accomplished using the label-free LC-MS discovery screen and ELISA based analysis of six nephrotoxicity biomarker proteins. Diagnostic performance of the expanded analyte set was statistically compared to conventional nephrotoxicity biomarkers. False discovery rate (FDR) analysis revealed 18 and 28 proteins from the CDDP and DOX groups, respectively, exhibiting significant differences between the vehicle and treated groups. Multiplex SRM assays were constructed to more precisely quantify candidate markers selected from the discovery screen and immunoassay experiments. To evaluate the sensitivity and specificity for each of the candidate biomarkers, histopathology severity scores were used as a benchmark for renal injury followed by receiver-operating characteristic (ROC) curve analysis on selected biomarkers. Further examination of the best performing analytes revealed relevant biological significance after consideration of anatomical localization and functional roles. In summary, the inclusion of mass spectrometry together with conventional ELISA based assays resulted in the identification of an expanded set of biomarkers with a realistic potential for providing additional beneficial information in mechanistic investigations of drug induced kidney injury and with similar responsiveness to conventionally applied indicators of renal injury.

Changes in the phosphoproteome of brown adipose tissue during hibernation in the ground squirrel, Ictidomys tridecemlineatus.

Mammalian hibernation is characterized by metabolic rate depression and a strong decrease in core body temperature that together create energy savings such that most species do not have to eat over the winter months. Brown adipose tissue (BAT), a thermogenic tissue that uses uncoupled mitochondrial respiration to generate heat instead of ATP, plays a major role in rewarming from deep torpor. In the present study we developed a label-free liquid chromatography mass spectrometry (LC-MS) strategy to investigate both differential protein expression and protein phosphorylation in BAT extracts from euthermic vs. hibernating ground squirrels (Ictidomys tridecemlineatus). In particular, we incorporated the filter-assisted sample preparation protocol, which provides a more in-depth analysis compared with gel-based and other LC-MS proteomics approaches. Surprisingly, mitochondrial membrane and matrix protein expression in BAT was largely constant between active euthermic squirrels and their hibernating counterparts. Validation by immunoblotting confirmed that the protein levels of mitochondrial respiratory chain complexes were largely unchanged in hibernating vs. euthermic animals. On the other hand, phosphoproteomics revealed that pyruvate dehydrogenase (PDH) phosphorylation increased during squirrel hibernation, confirmed by immunoblotting with phospho-specific antibodies. PDH phosphorylation leads to its inactivation, which suggests that BAT carbohydrate oxidation is inhibited during hibernation. Phosphorylation of hormone-sensitive lipase (HSL) was also found to increase during hibernation, suggesting that HSL would be active in BAT to produce the fatty acids that are likely the primary fuel for thermogenesis upon arousal. Increased perilipin phosphorylation along with that of a number of other proteins was also revealed, emphasizing the importance of protein phosphorylation as a regulatory mechanism during mammalian hibernation.

Global Proteome Changes in the Rat Diaphragm Induced by Endurance Exercise Training.

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, prolonged MV results in the rapid development of diaphragmatic atrophy and weakness. Importantly, endurance exercise training results in a diaphragmatic phenotype that is protected against ventilator-induced diaphragmatic atrophy and weakness. The mechanisms responsible for this exercise-induced protection against ventilator-induced diaphragmatic atrophy remain unknown. Therefore, to investigate exercise-induced changes in diaphragm muscle proteins, we compared the diaphragmatic proteome from sedentary and exercise-trained rats. Specifically, using label-free liquid chromatography-mass spectrometry, we performed a proteomics analysis of both soluble proteins and mitochondrial proteins isolated from diaphragm muscle. The total number of diaphragm proteins profiled in the soluble protein fraction and mitochondrial protein fraction were 813 and 732, respectively. Endurance exercise training significantly (P<0.05, FDR <10%) altered the abundance of 70 proteins in the soluble diaphragm proteome and 25 proteins of the mitochondrial proteome. In particular, key cytoprotective proteins that increased in relative abundance following exercise training included mitochondrial fission process 1 (Mtfp1; MTP18), 3-mercaptopyruvate sulfurtransferase (3MPST), microsomal glutathione S-transferase 3 (Mgst3; GST-III), and heat shock protein 70 kDa protein 1A/1B (HSP70). While these proteins are known to be cytoprotective in several cell types, the cyto-protective roles of these proteins have yet to be fully elucidated in diaphragm muscle fibers. Based upon these important findings, future experiments can now determine which of these diaphragmatic proteins are sufficient and/or required to promote exercise-induced protection against inactivity-induced muscle atrophy.

Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides.

Cephalosporin-3'-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms.

PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.

Probing the prostate tumour microenvironment I: impact of glucose deprivation on a cell model of prostate cancer progression.

In the developed world, prostate cancer is the most common cancer diagnosis in men. Although prostate cancer initially presents as a non life-threatening disease, 90% of patients will develop castration resistant prostate cancer (CRPC), which preludes distant metastasis and is largely accountable for prostate cancer associated deaths. This is because as yet, there are no viable molecular therapeutic targets for effective treatment of CRPC. It is now widely accepted that cancer cells can alter their metabolic profile during the course of tumourgenesis and metastasis such that they are able to survive in oxygen and nutrient-poor environments. This work was aimed towards gaining greater mechanistic understanding of how such stresses in the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under nutrient deficient (low glucose) conditions. This led to the identification of approximately 4,000 proteins - one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of low glucose conditions was established. Novel, intuitive workflows were subsequently implemented to ensure the verification of selected proteins of interest in a robust, reproducible and high throughput manner. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

Probing the prostate tumour microenvironment II: Impact of hypoxia on a cell model of prostate cancer progression

Approximately one in six men are diagnosed with Prostate Cancer every year in the Western world. Although it can be well managed and non-life threatening in the early stages, over time many patients cease to respond to treatment and develop castrate resistant prostate cancer (CRPC). CRPC represents a clinically challenging and lethal form of prostate cancer. Progression of CRPC is, in part, driven by the ability of cancer cells to alter their metabolic profile during the course of tumourgenesis and metastasis so that they can survive in oxygen and nutrient-poor environments and even withstand treatment. This work was carried out as a continuation of a study aimed towards gaining greater mechanistic understanding of how conditions within the tumour microenvironment impact on both androgen sensitive (LNCaP) and androgen independent (LNCaP-abl and LNCaP-abl-Hof) prostate cancer cell lines. Here we have applied technically robust and reproducible label-free liquid chromatography mass spectrometry analysis for comprehensive proteomic profiling of prostate cancer cell lines under hypoxic conditions. This led to the identification of over 4,000 proteins – one of the largest protein datasets for prostate cancer cell lines established to date. The biological and clinical significance of proteins showing a significant change in expression as result of hypoxic conditions was established. Novel, intuitive workflows were subsequently implemented to enable robust, reproducible and high throughput verification of selected proteins of interest. Overall, these data suggest that this strategy supports identification of protein biomarkers of prostate cancer progression and potential therapeutic targets for CRPC.

LC-MSE for Qualitative and Quantitative Proteomic Studies of Psychiatric Disorders.

This chapter describes the advantages and disadvantages of label-free liquid chromatography mass spectrometry in data-independent analysis mode (LC-MSE) in the identification of disease biomarkers in studies of psychiatric disorders. Along with the description of the technology, we discuss some of the most significant findings from various studies of post-mortem brain and neuroendocrine tissues from psychiatric disorder patients compared with controls. In addition, we describe some of the needs and challenges of performing these analyses in body fluids and peripheral tissues from living patients in order to increase translation of the findings into the clinical environment.

A quantitative proteomic screen of the Campylobacter jejuni flagellar-dependent secretome

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the world. A number of factors are believed to contribute to the ability of C. jejuni to cause disease within the human host including the secretion of non-flagellar proteins via the flagellar type III secretion system (FT3SS). Here for the first time we have utilised quantitative proteomics using stable isotope labelling by amino acids in cell culture (SILAC), and label-free liquid chromatography-mass spectrometry (LC/MS), to compare supernatant samples from C. jejuni M1 wild type and flagella-deficient (flgG mutant) strains to identify putative novel proteins secreted via the FT3SS. Genes encoding proteins that were candidates for flagellar secretion, derived from the LC/MS and SILAC datasets, were deleted. Infection of human CACO-2 tissue culture cells using these mutants resulted in the identification of novel genes required for interactions with these cells. This work has shown for the first time that both CJM1_0791 and CJM1_0395 are dependent on the flagellum for their presence in supernatants from C. jejuni stains M1 and 81–176. Biological significanceThis study provides the most complete description of the Campylobac er jejuni secretome to date. SILAC and label-free proteomics comparing mutants with or without flagella have resulted in the identification of two C. jejuni proteins that are dependent on flagella for their export from the bacterial cell.

Global Proteome Changes in Liver Tissue 6 Weeks after FOLFOX Treatment of Colorectal Cancer Liver Metastases.

(1) Oxaliplatin-based chemotherapy for colorectal cancer liver metastasis is associated with sinusoidal injury of liver parenchyma. The effects of oxaliplatin-induced liver injury on the protein level remain unknown. (2) Protein expression in liver tissue was analyzed—from eight patients treated with FOLFOX (combination of fluorouracil, leucovorin, and oxaliplatin) and seven controls—by label-free liquid chromatography mass spectrometry. Recursive feature elimination–support vector machine and Welch t-test were used to identify classifying and relevantly changed proteins, respectively. Resulting proteins were analyzed for associations with gene ontology categories and pathways. (3) A total of 5891 proteins were detected. A set of 184 (3.1%) proteins classified the groups with a 20% error rate, but relevant change was observed only in 55 (0.9%) proteins. The classifying proteins were associated with changes in DNA replication (p < 0.05) through upregulation of the minichromosome maintenance complex and with the innate immune response (p < 0.05). The importance of DNA replication changes was supported by the results of Welch t-test (p < 0.05). (4) Six weeks after FOLFOX treatment, less than 1% of identified proteins showed changes in expression associated with DNA replication, cell cycle entry, and innate immune response. We hypothesize that the changes remain after recovery from FOLFOX treatment injury.

Baseline Immunoglobulin E Levels as a Marker of Doxorubicin- and Trastuzumab-Associated Cardiac Dysfunction.

There is a critical need to develop robust, mechanistic strategies to identify patients at increased risk of cancer therapeutics-related cardiac dysfunction (CTRCD). We aimed to discover new biomarkers associated with doxorubicin- and trastuzumab-induced CTRCD using high-throughput proteomic profiling. Plasma, echocardiograms, and clinical outcomes were collected at standardized intervals in breast cancer patients undergoing doxorubicin and trastuzumab cancer therapy. Thirty-one longitudinal plasma samples from 3 cases with CTRCD and 4 age- and cancer-matched controls without CTRCD were processed and analyzed using label-free liquid chromatography-mass spectrometry. From these analyses, 862 proteins were identified from case/control pairs 1 and 2 and 1360 proteins from case/control pair 3. Proteins with a >1.5-fold change in cases compared with controls with a P<0.05 either at the time of CTRCD diagnosis or across all time points were considered candidate diagnostic or predictive biomarkers, respectively. The protein that demonstrated the largest differences between cases and controls was immunoglobulin E, with higher levels detected at baseline and across all time points in controls without CTRCD as compared with matched CTRCD cases (P<0.05). Similarly, in a validation study of 35 participants treated with doxorubicin and trastuzumab, high baseline immunoglobulin E levels were associated with a significantly lower risk of CTRCD (P=0.018). In patients receiving doxorubicin and trastuzumab, high baseline immunoglobulin E levels are associated with a lower risk of CTRCD. These novel findings suggest a new paradigm in cardio-oncology, implicating the immune system as a potential mediator of doxorubicin- and trastuzumab-induced cardiac dysfunction.

Molecular serum signature of treatment resistant depression.

A substantial number of patients suffering from major depressive disorder (MDD) do not respond to multiple trials of anti-depressants, develop a chronic course of disease and become treatment resistant. Most of the studies investigating molecular changes in treatment-resistant depression (TRD) have only examined a limited number of molecules and genes. Consequently, biomarkers associated with TRD are still lacking. This study aimed to use recently advanced high-throughput proteomic platforms to identify peripheral biomarkers of TRD defined by two staging models, the Thase and Rush staging model (TRM) and the Maudsley Staging Model (MSM). Serum collected from an inpatient cohort of 65 individuals suffering from MDD was analysed using two different mass spectrometric-based platforms, label-free liquid chromatography mass spectrometry (LC-MS(E)) and selective reaction monitoring (SRM), as well as a multiplex bead based assay. In the LC-MS(E) analysis, proteins involved in the acute phase response and complement activation and coagulation were significantly different between the staging groups in both models. In the multiplex bead-based assay analysis TNF-α levels (log(odds) = -4.95, p = 0.045) were significantly different in the TRM comparison. Using SRM, significant changes of three apolipoproteins A-I (β = 0.029, p = 0.035), M (β = -0.017, p = 0.009) and F (β = -0.031, p = 0.024) were associated with the TRM but not the MSM. Overall, our findings suggest that proteins, which are involved in immune and complement activation, may represent potential biomarkers that could be used by clinicians to identify high-risk patients. Nevertheless, given that the molecular changes between the staging groups were subtle, the results need to be interpreted cautiously.

Synaptic vesicle dynamic changes in a model of fragile X.

BackgroundFragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated.MethodsHere, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MSE) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy.ResultsKey findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission.ConclusionsOur findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-016-0080-1) contains supplementary material, which is available to authorized users.

Comparative Proteomics Analysis of Mouse Habu Nephritis Models with and without Unilateral Nephrectomy

Background/Aims: Individuals possessing a single kidney are at greater risk of renal injury upon exposure to harmful stimuli. This study aimed to explore the pathogenesis of renal injury in glomerulonephritis with versus without unilateral nephrectomy (UNX). Methods: Histological analysis and label-free quantitative proteomics were performed on two models—the Habu snake venom-induced glomerulonephritis model with versus without UNX (HabuU and Habu models, respectively). The role of villin 1, a differentially expressed protein (DEP) in mouse mesangial cells, was investigated. Results: Persistent mesangiolysis and focal hypercellularity together with reduced activation of cell proliferation in the HabuU model induced more serious renal injury compared with that in the Habu model. The DEPs between the two models were identified by label-free liquid chromatography-mass spectrometry. The KEGG pathway results indicated that regulation of actin cytoskeleton and focal adhesion were specifically enriched in the HabuU model. The cytoskeleton regulation protein villin 1 was downregulated in the HabuU model, but unchanged in the Habu model. Knockdown of villin 1 promoted apoptosis and inhibited the proliferation of mouse mesangial cells, suggesting villin 1 to be involved in qlomerular lesion self-repair insufficiency. Conclusion: By assessing the proteomic profiles of the two models, this study identified several important differences, particularly villin 1 expression, in regulatory mechanisms between the two models. Our findings provide novel insight into the mechanism of serious renal injury in glomerulonephritis with UNX.

Proteomics investigation identifies prominent changes in synapse-related proteins in a fragile X mouse model

Fragile X syndrome (FXS) is a single gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by trinucleotide repeats in the promoter region of the fragile X mental retardation gene (fmr1). This leads to the downregulation of the fragile X mental retardation protein (FMRP), which regulates translation of a wide range of proteins. The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated. Here, we have investigated the molecular mechanisms underlying FXS by identifying the molecular signature and elucidating the function of FXS-associated proteins. The first part of the study consists of an exploratory molecular profiling study on brain tissue samples of a fmr1 KO mouse model using shotgun label-free liquid chromatography mass spectrometry (LC-MS). These studies indicate that most of the changed proteins are involved in synaptic functions. Thus, as a next step we investigated synaptosomes from the cerebellum and hippocampus using LC-MS and label-based selected reaction monitoring (SRM). Key findings relate to altered levels of proteins involved in GABA-signalling in the cerebellum. To explore these findings further, FM1-43 dye was used in cultured hippocampal neurons and cerebellar Purkinje cells to track vesicle recycling and unloading profiles. Furthermore, ultrastructural analysis of synaptic vesicles of the GABA-ergic Purkinje cells in the cerebellum was performed to investigate potential pre-synaptic effects. Taken together, these studies provide novel insights into the molecular changes associated with FXS.