Comparative proteomics profile of osteoblasts cultured on dissimilar hydroxyapatite biomaterials: an iTRAQ-coupled 2-D LC-MS/MS analysis

Hydroxyapatite (HA) and its derived bioceramic materials have been widely used for skeletal implants and/or bone repair scaffolds. It has been reported that carbon nanotube (CNT) is able to enhance the brittle ceramic matrix without detrimental to the bioactivity. However, interaction between osteoblasts and these bioceramics, as well as the underlying mechanism of osteoblast proliferation on these bioceramic surfaces remain to be determined. Using iTRAQ-coupled 2-D LC-MS/MS analysis, we report the first comparative proteomics profiling of human osteoblast cells cultured on plane HA and CNT reinforced HA, respectively. Cytoskeletal proteins, metabolic enzymes, signaling, and cell growth proteins previous associated with cell adhesion and proliferation were found to be differentially expressed on these two surfaces. The level of these proteins was generally higher in cells adhered to HA surface, indicating a higher level of cellular proliferation in these cells. The significance of these findings was further assessed by Western blot analysis. The differential protein profile in HA and CNT strengthened HA established in our study should be valuable for future design of biocompatible ceramics.     

1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome

Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.     

iTRAQ-coupled 2D LC-MS/MS analysis on protein profile in vascular smooth muscle cells incubated with S- and R-enantiomers of propranolol: possible role of metabolic enzymes involved in cellular anabolism and antioxidant activity

Propranolol is a nonselective beta-blocker of the beta-adrenergic receptors, and the S-enantiomer is more active compared with the R-enantiomer. Clinically, it has been shown to be effective in hypermetabolic burn patients by decreasing cardiac work, protein catabolism, and lipolysis. While gene expression profiles have recently been reported in children receiving propranolol treatment, variations from one individual to another may have influenced the data analysis. Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the first study of protein profile in vascular smooth muscle cells incubated separately with the two enantiomers of propranolol. Four types of cellular proteins including metabolic enzymes, signaling molecules, cytoskeletal proteins, and those involved in DNA synthesis/protein translation displayed changes. The higher protein level of a number of enzymes involved in cellular anabolism and antioxidant activity in cells incubated with the S-enantiomer, as revealed by LC-MS/MS, was further supported by real-time PCR and Western blot analyses. Significantly, the increase in the anabolic activity associated with the higher level of metabolic enzymes was also supported by the higher intracellular concentration of the metabolic cofactor NAD+ which was a result of an increased oxidation of NADH. Our findings therefore provide molecular evidence on metabolic effect associated with propranolol treatment. The metabolic enzymes identified in our study may in turn be useful targets for future pharmaceutical interventions to reduce clinical side effects following propranolol treatment.     

Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS

Both organic solvent and surfactant have been used for dissolving membrane proteins for shotgun proteomics. In this work, two methods of protein solubilization, namely using 60% methanol or 1% SDS, to dissolve and analyze the inner membrane fraction of an Escherichia coli K12 cell lysate were compared. A total of 358 proteins (1417 unique peptides) from the methanol-solubilized protein mixture and 299 proteins (892 peptides) from the SDS-solubilized sample-were identified by using trypsin digestion and 2-D LC-ESI MS/MS. It was found that the methanol method detected more hydrophobic peptides, resulting in a greater number of proteins identified, than the SDS method. We found that 159 out of 358 proteins (44%) and 120 out of 299 proteins (40%) detected from the methanol- and SDS-solubilized samples, respectively, are integral membrane proteins. Among the 190 integral membrane proteins 70 were identified exclusively in the methanol-solubilized sample, 89 were identified by both methods, and only 31 proteins were exclusively identified by the SDS method. It is shown that the integral membrane proteins reflected the theoretical proteome for number of transmembrane helices, length, functional class, and topology, indicating there was no bias in the proteins identified.     

Enantioselective analysis of citalopram and demethylcitalopram in human and rat plasma by chiral LC-MS/MS: application to pharmacokinetics

Citalopram (CITA) is available as a racemic mixture and as a pure enantiomer. Its antidepressive action is related to the (+)-(S)-CITA and to the metabolite (+)-(S)-demethylcitalopram (DCITA). In the present investigation, a method for the analysis of CITA and DCITA enantiomers in human and rat plasma was developed and applied to the study of pharmacokinetics. Plasma samples (1 ml) were extracted at pH 9.0 with toluene:isoamyl alcohol (9:1, v/v). The CITA and DCITA enantiomers were analyzed by LC-MS/MS on a Chiralcel OD-R column. Recovery was higher than 70% for both enantiomers. The quantification limit was 0.1 ng/ml, and linearity was observed up to 500 ng/ml plasma for each CITA and DCITA enantiomer. The method was applied to the study of the kinetic disposition of CITA administered in a single oral dose of 20 mg to a healthy volunteer and in a single dose of 20 mg/kg (by gavage) to Wistar rats (n = 6 for each time). The results showed a higher proportion of the (-)-(R)-CITA in human and rat plasma, with S/R AUC ratios for CITA of 0.28 and 0.44, respectively. S/R AUC ratios of DCITA were 0.48 for rats and 1.04 for the healthy volunteer.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Saitohin Q7R polymorphism is associated with late‐onset Alzheimer's disease susceptibility among caucasian populations: a meta‐analysis

Saitohin (STH) Q7R polymorphism has been reported to influence the individual's susceptibility to Alzheimer's disease (AD); however, conclusions remain controversial. Therefore, we performed this meta‐analysis to explore the association between STH Q7R polymorphism and AD risk. Systematic literature searches were performed in the PubMed, Embase, Cochrane Library and Web of Science for studies published before 31 August 2016. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the strength of the association using a fixed‐ or random‐effects model. Subgroup analyses, Galbraith plot and sensitivity analyses were also performed. All statistical analyses were performed with STATA Version 12.0. A total of 19 case–control studies from 17 publications with 4387 cases and 3972 controls were included in our meta‐analysis. The results showed that the Q7R polymorphism was significantly associated with an increased risk of AD in a recessive model (RR versus QQ+QR, OR = 1.27, 95% CI = 1.01–1.60, P = 0.040). After excluding the four studies not carried out in caucasians, the overall association was unchanged in all comparison models. Further subgroup analyses stratified by the time of AD onset, and the quality of included studies provided statistical evidence of significant increased risk of AD in RR versus QQ+QR model only in late‐onset subjects (OR = 1.56, 95% CI = 1.07–2.26, P = 0.021) and in studies with high quality (OR = 1.37, 95% CI = 1.01–1.86, P = 0.043). This meta‐analysis suggests that the RR genotype in saitohin Q7R polymorphism may be a human‐specific risk factor for AD, especially among late‐onset AD subjects and caucasian populations.     

Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: Strategy for discovery of candidate serological biomarkers

In search of blood-based biomarkers that would enhance the ability to diagnose head and neck/oral squamous cell carcinoma (HNOSCC) in early stages or predict its prognosis, we analyzed the HNOSCC secretome (ensemble of proteins secreted and/or shed from the tumor cells) for potential biomarkers using proteomic technologies. LC-MS/MS was used to identify proteins in the conditioned media of four HNOSCC cell lines (SCC4, HSC2, SCC38, and AMOSIII); 140 unique proteins were identified on the basis of 5% global false discovery rate, 122 of which were secretory proteins, with 29 being previously reported to be overexpressed in HNOSCC in comparison to normal head and neck tissues. Of these, five proteins including α-enolase, peptidyl prolyl isomerase A/cyclophilin A, 14-3-3 ζ, heterogeneous ribonucleoprotein K, and 14-3-3 σ were detected in the sera of HNOSCC patients by Western blot analysis. Our study provides the evidence that analysis of head and neck cancer cells' secretome is a viable strategy for identifying candidate serological biomarkers for HNOSCC. In future, these biomarkers may be useful in predicting the likelihood of transformation of oral pre-malignant lesions, prognosis of HNOSCC patients and evaluate response to therapy using minimally invasive tests.     

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

HepG‐2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2‐D gel‐based and gel‐free methods. The analysis of crude HepG2 cell extracts by 2‐D DIGE provided data on 1835 protein spots which was then complemented by MS‐centered analysis of stable isotope labeling by amino acids in cell culture‐labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin‐induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin‐associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.     

LC-MS/MS测定小鼠血浆中多靶点抗AD bis-MEP-乙二酰胺杂合物ZLA浓度及药动学研究

目的:建立检测小鼠血浆内新型多靶点抗阿尔茨海默病(Alzheimer's disease,AD)药物双美普他酚-乙二酰胺杂合物(ZLA)浓度的高效液相色谱-质谱联用法(LC-MS/MS),并研究其在小鼠体内的药代动力学。方法:样品经甲醇沉淀去蛋白,应用Waters Xbridge C18色谱柱(2.1×100 mm,3.5μm),以甲醇-水(含5 m M甲酸铵,p H 9.8)(85:15,v/v)为流动相,流速0.25 m L/min;采用电喷雾(ESI)离子源,选择正离子模式多反应监测,待测物分别为m/z 304.3→107.0(ZLA)和m/z 621.7→232.1(内标)。分别给予KM小鼠腹腔和尾静脉注射ZLA 5mg/kg,不同时间点采集血浆用于ZLA定量分析。结果:ZLA和内标保留时间分别为3.2 min和2.5 min。血浆中ZLA线性范围为1-1000 ng/m L。血浆中提取回收率超过91%,日内和日间精密度RSD均小于6%。药动学研究结果显示,腹腔注射时ZLA可快速分布到血浆中,在10.2 min达到峰值,且能达到良好的生物利用度(47.6%)。结论:本研究建立的ZLA血药浓度测定方法快速、灵敏,特异性好,并成功应用于小鼠血浆中ZLA的药代动力学研究。本研究资料将为ZLA在AD治疗中的进一步临床前评估提供依据。     

Activation of PKA/SIRT1 signaling pathway by photobiomodulation therapy reduces Aβ levels in Alzheimer's disease models

A hallmark of Alzheimer's disease (AD) is the accumulation of amyloid‐β (Aβ), which correlates significantly with progressive cognitive deficits. Although photobiomodulation therapy (PBMT), as a novel noninvasive physiotherapy strategy, has been proposed to improve neuronal survival, decrease neuron loss, ameliorate dendritic atrophy, and provide overall AD improvement, it remains unknown whether and how this neuroprotective process affects Aβ levels. Here, we report that PBMT reduced Aβ production and plaque formation by shifting amyloid precursor protein (APP) processing toward the nonamyloidogenic pathway, thereby improving memory and cognitive ability in a mouse model of AD. More importantly, a pivotal protein, SIRT1, was involved in this process by specifically up‐regulating ADAM10 and down‐regulating BACE1, which is dependent on the cAMP/PKA pathway in APP/PS1 primary neurons and SH‐SY5Y cells stably expressing human APP Swedish mutation (APPswe). We further found that the activity of the mitochondrial photoacceptor cytochrome c oxidase (CcO) was responsible for PBMT‐induced activation of PKA and SIRT1. Together, our research suggests that PBMT as a viable therapeutic strategy has great potential value in improving cognitive ability and combatting AD.     

Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid-beta peptides via p75NTR/PLAIDD

One of the most important pathological features of Alzheimer's disease (AD) is extracellular senile plaques, whose major component is amyloid-beta peptides (Abeta). Abeta binds to the extracellular domain of p75NTR (p75 neurotrophin receptor) and induces neuronal cell death. We investigated the molecular mechanism of Abeta-induced neurotoxicity in detail from the standpoint of interaction between p75NTR and its recently identified relative, PLAIDD (p75-like apoptosis-inducing death domain). Using F11 neuronal hybrid cells, we demonstrate that there are two distinct pathways for Abeta-induced toxicity mediated by p75NTR. One pathway that has been previously elucidated, is mediated by p75NTR, Go, JNK, NADPH oxidase and caspase3-related caspases. We found that PLAIDD and Gi proteins, heterotrimeric G proteins, are involved in the alternative Abeta-induced neurotoxicity mediated by p75NTR. The alternative pathway triggered by Abeta is thus mediated by p75NTR, PLAIDD, Gi, JNK, NADPH oxidase and caspase3-related caspases. In addition, we found that HN, ADNF, IGF-I, or bFGF inhibits both pathways of Abeta-induced neurotoxicity mediated by p75NTR.     

Proteome characterization of splenocytes from an Aβpp/ps‐1 Alzheimer's disease model

Alzheimer's disease (AD) is the sixth leading cause of US deaths. In addition to neurodegenerative deficits in AD, changes in the immune system have also been observed. Proteomic analysis of specific immune cell populations may help gain insights into mechanisms of peripheral immunity in AD. Herein, we report proteome characterization for two subsets of splenocytes (i.e. CD90+ cells and a heterogeneous pool of CD90? cells) from a double transgenic mutant amyloid precursor protein/presenilin‐1 (Aβpp/ps‐ 1) AD mouse model. Overall, 906 proteins were identified from both cell types with 275 and 334 proteins uniquely identified as CD90+ and CD90? cells, respectively. Proteins identified in CD90+ and CD90? cells were significantly involved in 18 and 19 KEGG pathways, respectively. Amongst these, pathways associated with AD and antigen processing and presentation were identified in CD90+ and CD90? subsets, respectively. This is the first study to provide a reference proteome map for splenocyte populations in A βpp/ps‐ 1 double transgenic mice which will be helpful for future studies focused on understanding peripheral changes in this model. All MS data have been deposited in the ProteomeXchange with identifier PXD000203 ( http://proteomecentral.proteomexchange.org/dataset/PXD000203 ).     

Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ‐coupled 2‐D LC‐MS/MS analysis: Insights on liver angiogenesis

Hepatitis B virus (HBV) infection and in particular Hepatitis B Virus X Protein have been shown to modulate angiogenesis. However, a comprehensive and coordinated mechanism in the HBV‐induced angiogenesis remains to be established. In this study, transient transfection of replicative HBV genome was carried out in rat primary hepatocytes (RPHs) as well as HepG2 cells. Angiogenesis was assessed by tube formation assay. 2‐D LC‐MS/MS analysis was used to detect differentially expressed proteins in cells, supporting HBV replication compared with those transfected with the empty vector. A cell‐based HBV replication was established in both RPHs and HepG2 cells. HBV replication‐induced angiogenesis was indicated by tube formation of endothelial cells cultured in condition medium from RPHs or HepG2 cells supporting HBV replication. Enzymes associated with angiogenesis, namely fumarate hydratase and tryptophanyl‐tRNA synthetase, were identified by 2‐D LC‐MS/MS analysis in HBV replicating RPHs and HepG2 cells. Our results indicated that the application of quantitative proteomics based on iTRAQ can be an effective approach to evaluate the effects of HBV replication on liver angiogenesis. The angiogenesis‐associated proteins identified in our study may eventually lead to novel anti‐angiogenic hepatocellular carcinoma cancer therapy based on tumor vascular targeting or be the markers for hepatocellular carcinoma diagnosis.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.