Proteomic Analysis of Differential Protein Expression of Achilles Tendon in a Rabbit Model by Two-Dimensional Polyacrylamide Gel Electrophoresis at 21 Days Postoperation

Postoperative early kinesitherapy has been advocated as an optimal method for treating Achilles tendon rupture. However, an insight into the rationale of how early kinesitherapy contributes to healing of Achilles tendon remains to be achieved, and research in the area of proteomic analysis of Achilles tendon has so far been lacking. Forty-two rabbits were randomized into control group, immobilization group, and early motion group, and received postoperative cast immobilization and early motion treatments. Achilles tendon samples were prepared 21 days following microsurgery, and the proteins were separated with two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were first recognized by PDQuest software, and then identified using peptide mass fingerprinting, tandem mass spectrometry, and database searching. A total of 463 ± 12, 511 ± 39, and 513 ± 80 protein spots were successfully detected in the two-dimensional polyacrylamide gels for the Achilles tendon samples of rabbits in the control group, immobilization group, and early motion group, respectively. There were 15, 8, and 9 unique proteins in these three groups, respectively, and some differentially expressed proteins were also identified in each group. It was indicated that some of the differentially expressed proteins were involved in various metabolism pathways and may play an important role in healing of Achilles tendon rupture. Postoperative early kinesitherapy resulted in differentially expressed proteins in ruptured Achilles tendon compared with those treated with postoperative cast immobilization. These differentially expressed proteins may contribute to healing of Achilles tendon rupture through a mechanobiological mechanism due to the application of postoperative early kinesitherapy.     

Serum Proteomic Analysis of Cannabis Use Disorder in Male Patients

Cannabis use has been growing recently and it is legally consumed in many countries. Cannabis has a variety of phytochemicals including cannabinoids, which might impair the peripheral systems responses affecting inflammatory and immunological pathways. However, the exact signaling pathways that induce these effects need further understanding. The objective of this study is to investigate the serum proteomic profiling in patients diagnosed with cannabis use disorder (CUD) as compared with healthy control subjects. The novelty of our study is to highlight the differentially changes proteins in the serum of CUD patients. Certain proteins can be targeted in the future to attenuate the toxicological effects of cannabis. Blood samples were collected from 20 male individuals: 10 healthy controls and 10 CUD patients. An untargeted proteomic technique employing two-dimensional difference in gel electrophoresis coupled with mass spectrometry was employed in this study to assess the differentially expressed proteins. The proteomic analysis identified a total of 121 proteins that showed significant changes in protein expression between CUD patients (experimental group) and healthy individuals (control group). For instance, the serum expression of inactive tyrosine protein kinase PEAK1 and tumor necrosis factor alpha-induced protein 3 were increased in CUD group. In contrast, the serum expression of transthyretin and serotransferrin were reduced in CUD group. Among these proteins, 55 proteins were significantly upregulated and 66 proteins significantly downregulated in CUD patients as compared with healthy control group. Ingenuity pathway analysis (IPA) found that these differentially expressed proteins are linked to p38MAPK, interleukin 12 complex, nuclear factor-κB, and other signaling pathways. Our work indicates that the differentially expressed serum proteins between CUD and control groups are correlated to liver X receptor/retinoid X receptor (RXR), farnesoid X receptor/RXR activation, and acute phase response signaling.     

TMT-based proteomics analysis to screen potential biomarkers of Achyranthis Bidentatae Radix for osteoporosis in rats

This study aimed to explore the possible mechanism of Achyranthis Bidentatae Radix for the treatment of osteoporosis using tandem mass tag-based proteomics technique combined with mass spectrometry. Proteomics techniques combined with bioinformatics were used to analyze the biological functions of differentially expressed proteins. In addition, western blotting was performed to verify the expression of related proteins. A total of 3,752 proteins were identifiable by proteomic analysis. Furthermore, 93 differentially expressed proteins were identified, of which 61 were upregulated and 32 were downregulated. Differentially expressed proteins were primarily associated with oxidative phosphorylation (p = 4.8 × 10⁻⁴) pathways and involved in transmembrane transport (p = 3.5 × 10⁻³), exocytic process (p = 1.2 × 10⁻²), cellular developmental process (p = 1.3 × 10⁻²), adenosine triphosphate metabolic process (p = 1.0 × 10⁻²) and other biological processes. Western blotting analysis showed that MT-CYB and NDUFA9 were differentially expressed in the bone microenvironment of rats with osteoporosis. We speculated that they were potential biomarkers linked to osteoporosis. This study employed proteomics to explore the potential therapeutic targets of Achyranthis Bidentatae Radix to treat osteoporosis. This revealed that mitochondria are a new target for the treatment of glucocorticoid-induced osteoporosis.     

Changes in amniotic fluid and umbilical cord serum proteomic profiles of foetuses with intrauterine growth retardation

Foetal growth is a result of a complex net of processes, requiring coordination within the maternal, placental, and foetal compartments, the imbalance or lack of which may lead to intrauterine growth restriction (IUGR). IUGR is the major cause of perinatal morbidity and mortality, and is also related to enhanced morbidity and metabolic abnormalities later in life. In the present study, the protein profiles of umbilical cord serum (UCS) and amniotic fluid (AF) of ten IUGR and ten appropriate for gestational age newborns have been analysed by 2-DE, and nanoHPLC-Chip/MS technology. A total of 18 and 13 spots were found to be differentially expressed (p<0.01) in UCS and AF respectively. The unique differentially expressed proteins identified by MS/MS analysis were 14 in UCS, and 11 in AF samples. Protein gene ontology classification indicate that 21% of proteins are involved in inflammatory response, 20% in immune response, while a smaller proportion are related to transport, blood pressure, and coagulation. These results support the conclusion that the IUGR condition alters the expression of proteins involved in the coagulation process, immune mechanisms, blood pressure and iron and copper homeostasis control, offering a new insight into IUGR pathogenesis.     

Proteomic Analysis of Differentially Expressed Proteins of Arabidopsis thaliana Response to Specialist Herbivore Plutella xylostella

The changes of the proteome in Arabidopsis thaliana leaves were examined by specialist Plutella xylostella.Analysis of about 1100 protein spots on each 2DE gel revealed 38 differentially expressed protein spots in abun-dance of which 34 proteins were identified by MALDI-TOF/TOF MS.Among the insect feeding responsive proteins,a few proteins involved in carbon metabolism were identified including proteins associated with the Calvin cycle in the chloroplast and TCA cycle in the mitochondria,indicating carbon metabolism related proteins may play crucial roles in induced defense response in plants under insect infestation.The analysis elucidates the subcellular location of proteins demonstrates that about 50% of proteins are in the chloroplast,which shows the chloroplast has a key role in the insect feeding response for plant.Gene expression analysis of 10 different proteins by quantitative real-time PCR shows that four proteins of the mRNA level were correlated well with the protein level.This study further dissected the nature of insect infestation as a stress signal and some novel insect feeding responsive proteins identified may play an important role in induced defence machanism for plant.     

不稳定性心绞痛血瘀证的血浆蛋白质组学研究

为了寻找冠心病不稳定性心绞痛血瘀证血浆差异表达蛋白, 探索冠心病不稳定性心绞痛血瘀证的蛋白质组学特点. 采用差异凝胶双向电泳和质谱联用技术对12例冠心病不稳定性心绞痛血瘀证患者和12例健康人血浆进行比较研究. 初步发现了Fibrinogen β chain, Fibrinogen γ chain, α1-Antitrypsin, Haptoglobin β chain, Haptoglobin α2 chain在冠心病不稳定性心绞痛血瘀证患者中高表达, ApoA-IV, ApoA-I, Transthyretin, ApoJ在冠心病不稳定性心绞痛血瘀证患者中低表达. 差异表达蛋白根据功能可分为以下三类: (1)急性时相反应负相蛋白; (2)载脂蛋白; (3)凝血相关蛋白. 冠心病不稳定性心绞痛血瘀证可能与炎症反应、脂代谢紊乱以及凝血功能异常相关.     

Quantitative proteomic analysis of serum proteins in patients with Parkinson's disease using an isobaric tag for relative and absolute quantification labeling, two-dimensional liquid chromatography, and tandem mass spectrometry

Parkinson's disease (PD) is a common disease which occurs in aged people with chronic, progressive degenerative character of the central nervous system. Until now there is no effective treatment method in PD patients before they show obvious symptoms for prevention and early diagnosis. In order to find out early disease specific biomarkers, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the different disease progress types of PD. 26 proteins were differentially expressed in a total of 258 identified proteins by proteomic techniques. The expression level of eight proteins which included sero-transferrin and clusterin increased. The expression level of eighteen proteins which include complement component 4B, apolipoprotein A-I, α-2-antiplasmin and coagulation factor V decreased. Those proteins may be associated with oxidative stress, mitochondrial dysfunction, abnormal protein aggregation and inflammation. In this study, the expression level of apolipoprotein A-I decreased, particularly in the early stage of PD patients. This protein regulated not only the lipid metabolism in the central nervous system, but also influenced the deposition process of proteins which are involved in neural degenerative diseases, such as the pathogenesis of PD.     

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.     

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.     

嗅觉记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了C57BL/6J小鼠在嗅觉训练和记忆测试后嗅球蛋白表达的差异, 探讨了与嗅觉记忆相关的蛋白质. C57BL/6J小鼠经嗅觉训练后, 可对相应的气味保持记忆能力, 其嗅球蛋白表达存在明显差异, 5种蛋白与嗅觉记忆形成显著相关. 上述蛋白功能涉及神经发育, 信号转导及细胞骨架和核苷酸代谢, 其中神经发育和信号转导相关蛋白表达上调, 而细胞骨架和核苷酸代谢相关蛋白表达水平降低. 这些与嗅觉记忆形成相关的蛋白深化了对嗅觉记忆机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

Understanding the mechanisms of cancers based on function sub-pathways

Pathway analysis has become a popular technology tool for gaining insight into the underlying biology of differentially expressed genes and proteins. Although many sub-pathways analysis methods have been proposed, the function of these sub-pathways is generally implicit. In this paper, we propose a function sub-pathway analysis (FSPA) method which includes all nodes reaching a specific function node at the downstream of pathways. The perturbation degree of a sub-pathway is defined as the negative of the log p-value of the sub-pathway. The proposed FSPA allows analyzing the differentially expressed genes in a sub-pathway with diseases in explicit function level. Results from six datasets of colorectal cancer, lung cancer and pancreatic cancer show that the proposed FSPA could identify more cancer associated pathways. And more importantly, it could identify which sub-pathways lead to a specific abnormal function, and to what extent it affects the function. Furthermore, the proposed perturbation degree could also analyze the imbalance of some functions involved in some biological process. The results by FSPA are helpful for elucidating the underlying mechanisms of cancers and designing therapeutic strategies.     

Down-regulation of Vinculin in Human Colorectal Carcinoma Identified by Proteomics Analysis

In the present study, we aimed to globally profile the proteins involved in colorectal carcinoma(CRC), in order to find clues to the pathological process of CRC. Pairs of malignant tissues and their adjacent healthy tissues from patients with colorectal cancer were subject to differential proteomics analysis. Two dimensional electrophoresis coupled with mass spectrometry(2-DE/MS) was used to identify differentially expressed proteins between pairs of tissue samples. A list of proteins relevant to the progression of colorectal tumor was identified by two dimensional gel electrophoresis(2-DE)-based proteomics approach. Among the identified proteins, vinculin was found to be remarkably down-regulated in colorectal carcinoma tissues. In addition, three phosphorylation modifications within the isolated vinculin were identified by in-depth liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis. Our results provide a basis for further understanding the pathological significance of vinculin in the regulation of carcinogenesis, invasion and metastasis of colorectal tumors.     

Proteomic analysis reveals metabolic changes during yeast to hypha transition in Yarrowia lipolytica

Fungal dimorphism is important for survival in different environments and has been related to virulence. The ascomycete Yarrowia lipolytica can grow as yeast, pseudomycelial or mycelial forms. We have used a Y. lipolytica parental strain and a Deltahoy1 mutant, which is unable to form hypha, to set up a model for dimorphism and to characterize in more depth the yeast to hypha transition by proteomic techniques. A two-dimensional gel electrophoresis (2-DE) based differential expression analysis of Y. lipolytica yeast and hyphal cells was performed, and 45 differentially expressed proteins were detected; nine with decreased expression in hyphal cells were identified. They corresponded to the S. cerevisiae homologues of Imd4p, Pdx3p, Cdc19, Sse1p, Sol3p, Sod2p, Xpt1p, Mdh1p and to the unknown protein YALIOB00924g. Remarkably, most of these proteins are involved in metabolic pathways, with four showing oxidoreductase activity. Furthermore, taking into account that this is the first report of 2-DE analysis of Y. lipolytica protein extracts, 35 more proteins from the 2D map of soluble yeast proteins, which were involved in metabolism, cell rescue, energy and protein synthesis, were identified.     

蛋白质组学技术筛选与鉴定癫痫疾病的差异蛋白质

应用蛋白质组学双向凝胶电泳(Two-dimensional gel electrophoresis, 2DE)和质谱技术, 定量分析和鉴定了癫痫组(n=3)和正常组(n=3)脑组织的差异表达蛋白, 以从蛋白质水平上揭示癫痫病的发机制. 结果表明, 凝胶图谱可辨识2500~3000个蛋白点, 对21个显著差异表达蛋白点进行质谱鉴定和SwissProt数据库检索, 得到17个癫痫差异蛋白, 其中2个蛋白在癫痫组织中表达上调, 15个蛋白表达下调. 部分蛋白与癫痫的关系属首次报道. 这些蛋白与癫痫的发生发展相关, 可能成为癫痫的分子标志物和药物治疗的靶向蛋白.     

Comprehensive Analysis of the Effect of 20(R)-Ginsenoside Rg3 on Stroke Recovery in Rats via the Integrative miRNA–mRNA Regulatory Network

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNAs. Recent research has proven that miRNAs play an essential role in the occurrence and development of ischemic stroke. Our previous studies confirmed that 20(R)-ginsenosideRg3 [20(R)-Rg3] exerts beneficial effects on cerebral ischemia–reperfusion injury (CIRI), but its molecular mechanism has not been elucidated. In this study, we used high-throughput sequencing to investigate the differentially expressed miRNA and mRNA expression profiles of 20(R)-Rg3 preconditioning to ameliorate CIRI injury in rats and to reveal its potential neuroprotective molecular mechanism. The results show that 20(R)-Rg3 alleviated neurobehavioral dysfunction in MCAO/R-treated rats. Among these mRNAs, 953 mRNAs were significantly upregulated and 2602 mRNAs were downregulated in the model group versus the sham group, whereas 437 mRNAs were significantly upregulated and 35 mRNAs were downregulated in the 20(R)-Rg3 group in contrast with those in the model group. Meanwhile, the expression profile of the miRNAs showed that a total of 283 differentially expressed miRNAs were identified, of which 142 miRNAs were significantly upregulated and 141 miRNAs were downregulated in the model group compared with the sham group, whereas 34 miRNAs were differentially expressed in the 20(R)-Rg3 treatment group compared with the model group, with 28 miRNAs being significantly upregulated and six miRNAs being significantly downregulated. Furthermore, 415 (391 upregulated and 24 downregulated) differentially expressed mRNAs and 22 (17 upregulated and 5 downregulated) differentially expressed miRNAs were identified to be related to 20(R)-Rg3′s neuroprotective effect on stroke recovery. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that 20(R)-Rg3 could modulate multiple signaling pathways related to these differential miRNAs, such as the cGMP-PKG, cAMP and MAPK signaling pathways. This study provides new insights into the protective mechanism of 20(R)-Rg3 against CIRI, and the mechanism may be partly associated with the regulation of brain miRNA expression and its target signaling pathways.     

Proteomic analysis reveals changes in the hippocampus protein pattern of rats exposed to dietary zinc deficiency

An experiment was performed to observe protein changes in the hippocampus of zinc‐deficient (ZD) rats. Twenty‐four male weanling Wistar rats were randomly assigned to ZD (n=12) and control groups (n=12). After 4‐wk treatment, we used 2‐DE and MALDI‐TOF‐MS to analyze the proteomes of hippocampus in the two groups. One of the important differential proteins, ubiquitin C‐terminal hydrolase L1 (Uch‐L1), was confirmed by Western blot assays. The results demonstrated that compared with the controls, ZD rats had significantly reduced plasma zinc concentration and alkaline phosphatase activity. The latency period in passive avoidance performance was also significantly shorter for the ZD rats. Nine proteins were differentially expressed between the two groups. Eight of them were identified. Tubulin β chain and voltage‐dependent anion channel 1 were upregulated, while mitochondrial aldehyde dehydrogenase, α‐enolase, dimethylarginine dimethylaminohydrolase 1, F‐actin capping protein α‐2 subunit, pyruvate dehydrogenase β and Uch‐L1 were downregulated, respectively. Importantly, some of the identified proteins (e.g. Uch‐L1) are known to be involved in cognitive impairment. Western blot analysis of hippocampus Uch‐L1 expression confirmed the proteomic findings. The data indicated that there may be common mechanisms or pathways in cognitive dysfunction between neurodegenerative diseases and zinc deficiency.     

Comparative proteomic analysis of different Toxoplasma gondii genotypes by two‐dimensional fluorescence difference gel electrophoresis combined with mass spectrometry

Toxoplasma gondii is a protozoan parasite infecting almost all warm‐blooded animals and humans. There are three infective stages of T. gondii: the tachyzoites, the bradyzoites, and the oocysts. The tachyzoite is a rapidly multiplying stage and the main pathogenic factor. In North America and Europe, T. gondii is consisted of four major clonal lineages (namely Types I, II, III, and Type 12). In this study, we explored the proteomic profiles of different genotypes (Type I‐RH strain, Type II‐PRU strain, Type II‐TgQHO strain, and ToxoDB 9‐TgC7 strain) of T. gondii tachyzoites by using 2D DIGE combined with MALDI‐TOF MS. Totally, 110 differentially abundant protein spots were selected. Of these, 98 spots corresponding to 56 proteins from T. gondii were successfully identified. These included surface antigen (SAG1), heat shock protein 70 (Hsp 70), disulfide isomerase, coronin, heat shock protein 60 (Hsp 60), pyruvate kinase, receptor for activated C kinase 1, and peroxiredoxin. Gene ontology enrichment analysis revealed that most of the differentially abundant proteins were involved in biological regulation, metabolic process, response to stress, binding, antioxidant activity, and transporter activity. According to the KEGG metabolic pathway maps of T. gondii, some identified proteins were involved in the glycolytic/gluconeogenesis pathway. The present study identified differentially abundant proteins among different genotypes of T. gondii and these findings have implications for the better understanding of the phenotypic differences among the examined T. gondii genotypes, which in turn may contribute to the better control of toxoplasmosis.     

快速老化模型小鼠海马蛋白质组学初步研究

应用双向凝胶电泳结合质谱鉴定, 分析比较6月龄和12月龄快速老化模型小鼠(Senescence-accele-rated mouse, SAM)的快速老化亚系SAM-prone/8(SAMP8)及抗快速老化亚系SAM-resistance/1(SAMR1)海马蛋白表达的差异, 从蛋白质水平初步探讨与老化相关的学习记忆功能障碍的发生机制. 结果表明, 与同龄SAMR1比较, 6月龄SAMP8海马中有15个蛋白点表达显著上调, 5个蛋白点表达显著下调; 12月龄SAMP8海马中有12个蛋白点表达显著上调, 2个蛋白点表达显著下调, 2个蛋白点只在SAMP8中有表达. 应用质谱分析结合数据库检索, 共鉴定了22种蛋白质. 6月龄和12月龄SAMP8与SAMR1海马中表达有明显变化的蛋白按功能可分为如下4类: (1) 能量代谢相关蛋白; (2) 线粒体功能相关蛋白; (3) 信号转导相关蛋白; (4) 其它蛋白. 研究结果表明, SAMP8和SAMR1海马蛋白表达存在明显差异, 其中一些蛋白与SAMP8随龄出现的学习记忆功能减退相关, 并可能为研究或发现促智药物作用的新蛋白靶标提供线索.