Label-free Liquid Chromatography Research Articles

Proteomic profiling of the plasma of Gambian children with cerebral malaria

BackgroundCerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection. A number of pathological findings have been correlated with pediatric CM including sequestration, platelet accumulation, petechial haemorrhage and retinopathy. However, the molecular mechanisms leading to death in CM are not yet fully understood.MethodsA shotgun plasma proteomic study was conducted using samples form 52 Gambian children with CM admitted to hospital. Based on clinical outcome, children were assigned to two groups: reversible and fatal CM. Label-free liquid chromatography–tandem mass spectrometry was used to identify and compare plasma proteins that were differentially regulated in children who recovered from CM and those who died. Candidate biomarkers were validated using enzyme immunoassays.ResultsThe plasma proteomic signature of children with CM identified 266 proteins differentially regulated in children with fatal CM. Proteins from the coagulation cascade were consistently decreased in fatal CM, whereas the plasma proteomic signature associated with fatal CM underscored the importance of endothelial activation, tissue damage, inflammation, haemolysis and glucose metabolism. The concentration of circulating proteasomes or PSMB9 in plasma was not significantly different in fatal CM when compared with survivors. Plasma PSMB9 concentration was higher in patients who presented with seizures and was significantly correlated with the number of seizures observed in patients with CM during admission.ConclusionsThe results indicate that increased tissue damage and hypercoagulability may play an important role in fatal CM. The diagnostic value of this molecular signature to identify children at high risk of dying to optimize patient referral practices should be validated prospectively.

The influence of physiological environment on the targeting effect of aptamer-guided gold nanoparticles

Aptamer guided nanomedicine shows great promise in targeted cancer therapies. However the loss of targeting capacity during in vivo or clinical trials has largely hindered its popularity and there are no systematic studies to elucidate the causes. Herein, we investigated such loss of targeting capacity by examining how the physiological milieu affected targeting effect. Aptamer functionalized gold nanoparticle (AuNP) was chosen as the model and exposed to human blood serum that is used to mimic physiological milieu. Dynamic light scattering (DLS), flow cytometry and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to determine variations of NPs’ surface chemistry and biological identities changes after serum exposure. Results showed that the targeting ability loss was caused by protein corona blocking, replacement and enzymatic cleavage of surface aptamer targeting ligands. Noteworthy, the aggregation issue is critical for the smaller NPs. Analysis of the protein corona profile indicated the accumulation of immune-related proteins on the surface of aptamer-conjugated NPs, which could induce immune response, resulting in rapid clearance of NPs.

Extracellular vesicles from pluripotent stem cell-derived mesenchymal stem cells acquire a stromal modulatory proteomic pattern during differentiation

Mesenchymal stem/stromal cells (MSCs) obtained from pluripotent stem cells (PSCs) constitute an interesting alternative to classical MSCs in regenerative medicine. Among their many mechanisms of action, MSC extracellular vesicles (EVs) are a potential suitable substitute for MSCs in future cell-free-based therapeutic approaches. Unlike cells, EVs do not elicit acute immune rejection, and they can be produced in large quantities and stored until ready to use. Although the therapeutic potential of MSC EVs has already been proven, a thorough characterization of MSC EVs is lacking. In this work, we used a label-free liquid chromatography tandem mass spectrometry proteomic approach to identify the most abundant proteins in EVs that are secreted from MSCs derived from PSCs (PD-MSCs) and from their parental induced PSCs (iPSCs). Next, we compared both datasets and found that while iPSC EVs enclose proteins that modulate RNA and microRNA stability and protein sorting, PD-MSC EVs are rich in proteins that organize extracellular matrix, regulate locomotion, and influence cell–substrate adhesion. Moreover, compared to their respective cells, iPSCs and iPSC EVs share a greater proportion of proteins, while the PD-MSC proteome appears to be more specific. Correlation and principal component analysis consistently aggregate iPSCs and iPSC EVs but segregate PD-MSC and their EVs. Altogether, these findings suggest that during differentiation, compared with their parental iPSC EVs, PD-MSC EVs acquire a more specific set of proteins; arguably, this difference might confer their therapeutic properties.

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.

Downregulation of tight junction protein zonula occludens-2 and urothelium damage in a cyclophosphamide-induced mouse model of cystitis

ObjectivesThe cyclophosphamide (CYP)-induced model of cystitis in mice closely fits the symptoms of chronic bladder inflammation. Cystitis was recently found to be due to an altered gap junction protein in a rat model. Thus, this study was conducted to evaluate changes in protein expression and composition in the bladder of CYP-treated mice. Materials and methodsAdministration of CYP induced cystitis-related symptoms in mice. Cystometry was assessed and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured. Voiding interval values (time between voids) were assessed in mice under anesthesia. The bladders were removed for proteomic analysis using label-free quantitative proteomics and liquid chromatography–mass spectrometry. Additionally, immunochemistry (IHC) and Western blot were used to confirm the location and level, respectively, of ZO-2 expression. ResultsCompared to the control group, the voiding interval values and urothelial thickness in the bladder in the CYP-treated group were significantly decreased. Additionally, we identified 105 differentially expressed proteins in the bladder of CYP-treated mice with proteomic analysis. These proteins were involved in cell–cell tight junctions, exocytosis, muscle development, contraction, and regulation, immune responses, proteolysis, and cell adhesion. IHC and Western blot confirmed the downregulation of the tight junction protein ZO-2 in the urothelium of bladder. ConclusionsOur results suggest that downregulation in tight junction protein ZO-2 and urothelium damage may have a role in cystitis-related OAB. These changes could be related to the molecular mechanism of cystitis-related OAB.

Method for Label-Free Quantitative Proteomics for Sorghum bicolor L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.

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.

P.240 - Proteomic identification of novel brain and serum biomarkers linked to the pathophysiology of Duchenne muscular dystrophy

Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood, and is characterised by primary abnormalities in the Dmd gene encoding the membrane cytoskeletal protein dystrophin. Aside from the well-established skeletal muscle degeneration and cardio-respiratory complications, extra-muscular manifestations are also evident. Comparative label-free liquid chromatography mass spectrometric analyses of brain tissue and serum derived from the mdx-4cv animal model of the disease have identified novel proteomic markers of astrogliosis and inflammation respectively. The elucidation of proteome-wide changes in the mdx-4cv dystrophin-deficient brain revealed alterations in proteins involved in cytoskeletal stabilisation, calcium homeostasis, metabolism and reactive gliosis. Elevated abundance of some brain-associated proteins were also detected in serum samples, suggesting that the presence of brain-derived proteins in serum could potentially reflect the extent of dystrophinopathy in the brain. A large number of differentially abundant proteins in immunodepleted mdx-4cv serum were also identified by label-free mass spectrometry. In particular these proteins were associated with chronic tissue damage, necrosis and sterile inflammation. A variety of these potential circulatory biomarkers linked to muscular dystrophy were validated independently by comparative immunoblot analysis and enzyme-linked immunosorbent assays. Further research will be required to investigate whether such protein biomarkers are passively leaked or actively secreted into the circulatory system. These novel protein marker candidates identified by proteomic profiling of brain tissue and serum offer insights into the pathophysiology of dystrophinopathy and may be further investigated for their potential use in the diagnosis, prognosis and therapeutic monitoring of Duchenne muscular dystrophy.

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.

β-catenin downregulates Dicer to promote ovarian cancer metastasis.

Ovarian cancer is a nearly uniform lethal disease and its highly aggressive metastatic phenotype portends a poor prognosis. Lack of a well-controlled, relevant experimental model has been a major obstacle to identifying key molecules causing metastasis. Here we describe the creation of a new isogenic model of spontaneous human ovarian cancer metastasis exhibiting opposite phenotypes-highly metastatic (HM) and non-metastatic (NM)-both in vitro and in vivo. HM was unique in its ability to metastasize consistently to the peritoneum, mimicking the major dissemination route of human ovarian cancer. In contrast, NM failed to form detectable metastases, although it was equally tumorigenic. Using comparative label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified β-catenin, which we demonstrated for the first time as having a direct role in the pathogenesis of ovarian cancer metastasis. Our studies also revealed a previously unrecognized role of β-catenin in the downregulation of multiple microRNAs (miRNAs) through attenuating miRNA biogenesis by targeting Dicer, a key component of the miRNA-processing machinery. One such downregulated miRNAs was miR-29s involved in epithelial-to-mesenchymal transition and subsequent stem cell traits. Silencing β-catenin or overexpressing Dicer or miR-29 mimics in HM significantly reduced the ability of these cells to migrate. β-catenin-knockdown cells also failed to metastasize in an orthotopic model of ovarian cancer. Meta-analysis revealed an increase in CTNNB1 and a decrease in DICER1 expression levels in the high-risk group. These results uncover β-catenin as a critical factor in promoting ovarian cancer aggressiveness and a new mechanism linking between β-catenin and miRNA downregulation underlying this process.

A promising protein responsible for overactive bladder in ovariectomized mice

ObjectiveOvariectomy (OVX) in mice is a model mimicking a neuro-electronic proof of an overactive bladder in postmenopausal women. Overactive bladder (OAB) was recently found to be due to an altered gap junction protein in a rat model. Thus, this study was conducted to evaluate changes in cell junction protein expression and composition in the bladder of OVX mice. Materials and MethodsThirty-six virgin female mice were randomized into three groups: mice with a sham operation only (control), OVX mice without estradiol (E2) replacement, and OVX mice with E2 replacement (OVX + E2). Cystometry assessment was conducted and cell junction-associated protein zonula occludens-2 (ZO-2) expression was measured after 8 weeks. Voiding interval values (time between voids) were assessed in mice under anesthesia. After measurements, the bladders were removed for proteomic analysis using the label-free quantitative proteomics and liquid chromatography–mass spectrometry technology. Lastly, immunohistochemistry (IHC) and Western blot were used to confirm the location and level, respectively, of ZO-2 expression. ResultsWe identified 73 differentially expressed proteins in the bladder of OVX mice. The OVX mice showed significantly lower voiding interval values. Voiding interval values were significantly higher in the OVX + E2 group than in the OVX group. Urothelial thicknesses in the bladder were also significantly lower in the OVX group than in the control group. E2 replacement reversed the urothelium layers. Additionally, the expression of ZO-2, a tight junction protein, was the most affected by OVX treatment. IHC and Western blot confirmed the downregulation of ZO-2 in the bladder of OVX mice. Expression of ZO-2 protein was significantly increased in OVX + E2 group compared with OVX group. ConclusionThis exploratory study estimated changes in protein expression and composition in the bladder of OVX mice. These changes may be associated with the molecular mechanisms of OAB.

Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. features. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties’ SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

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.

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.

Label-free proteomic analysis of placental proteins during Toxoplasma gondii infection

Toxoplasma gondii is a ubiquitous, obligate intracellular parasite capable of crossing the placental barrier and causing spontaneous abortion, preterm labor, or significant disease in the surviving neonate. To better understand molecular mechanisms underlying abnormal pregnancy outcomes caused by T. gondii, placental proteins extracted from T. gondii-infected and -uninfected mice were comparatively analyzed using label-free liquid chromatography–tandem mass spectrometry. Significant difference was observed in the expression of 58 out of 792 proteins in infected placentas (p<0.05) compared with that in uninfected placentas. Quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining were used to validate the results of the proteomic analysis. Some placental proteins differentially expressed in infected and uninfected mice were found to be associated with several different biological processes of pregnancy, particularly with trophoblast invasion and placental development. The results provide possible novel insights into the molecular mechanisms for abnormal pregnancy outcomes associated with T. gondii infection. SignificanceIn order to further explore the mechanisms of abnormal pregnant outcomes caused by T. gondii infection, we first applied label-free proteomic technology to analyze the differentially expressed host placental proteins with T. gondii infection. The results showed that some differential proteins are associated with trophoblast invasion and placenta development. The findings provide a systemic view of the altered placental proteins and help to declare the molecular mechanisms of abnormal pregnancy outcomes caused by T. gondii infection.

Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients.

There is unmet need for prediction of treatment response for chronic myeloid leukemia (CML) patients. The present study aims to identify disease-specific/disease-associated protein biomarkers detectable in bone marrow and peripheral blood for objective prediction of individual’s best treatment options and prognostic monitoring of CML patients. Bone marrow plasma (BMP) and peripheral blood plasma (PBP) samples from newly-diagnosed chronic-phase CML patients were subjected to expression-proteomics using quantitative two-dimensional gel electrophoresis (2-DE) and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of 2-DE protein fingerprints preceding therapy commencement accurately predicts 13 individuals that achieved major molecular response (MMR) at 6 months from 12 subjects without MMR (No-MMR). Results were independently validated using LC-MS/MS analysis of BMP and PBP from patients that have more than 24 months followed-up. One hundred and sixty-four and 138 proteins with significant differential expression profiles were identified from PBP and BMP, respectively and only 54 proteins overlap between the two datasets. The protein panels also discriminates accurately patients that stay on imatinib treatment from patients ultimately needing alternative treatment. Among the identified proteins are TYRO3, a member of TAM family of receptor tyrosine kinases (RTKs), the S100A8, and MYC and all of which have been implicated in CML. Our findings indicate analyses of a panel of protein signatures is capable of objective prediction of molecular response and therapy choice for CML patients at diagnosis as ‘personalized-medicine-model’.

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.