基于iTRAQ技术结合2D-LC-MS/MS研究黄连对CYP450同工酶表达的影响

黄连是临床常用药材,在中医药中被广泛应用,具有清热燥湿、泻火解毒之功,中药成分比较复杂,细胞色素P450(cytochrome P450,CYP450)同工酶是药物的主要代谢酶,研究黄连对CYP450酶的影响具有很重要的临床意义。实验选用大鼠肝脏组织,差速离心法分离肝微粒体,BCA(bicinchoninic acid)蛋白定量,应用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)技术结合2D-LC-MS/MS筛选差异蛋白。黄连一共鉴定出30个CYP450同工酶,7个表达上调,8个表达下调,15个没有发生变化。iTRAQ技术结合2D-LC-MS/MS可以全面研究CYP450酶,但黄连有必要进一步在体外水平对其进行评价,预防潜在的临床药物相互作用。     

Identification of GlcNAcylated alpha-1-antichymotrypsin as an early biomarker in human non-small-cell lung cancer by quantitative proteomic analysis with two lectins

Background:

Non-small-cell lung cancer (NSCLC) is the main type of lung cancer with high mortality rates in worldwide. There is a need to identify better biomarkers to detect NSCLC at an early stage as this will improve therapeutic effect and patient survival rates.

Methods:

Two lectins (AAL/AAGL and AAL2/AANL), which specifically bind to tumour-related glycan antigens, were first used to enrich serum glycoproteins from the serum of early NSCLC patients, benign lung diseases subjects and healthy individuals. The samples were investigated by using iTRAQ labelling and LC-MS/MS.

Results:

A total of 53 differentially expressed proteins were identified by quantitative proteomics and four glycoproteins (AACT, AGP1, CFB and HPX) were selected for further verification by western blotting. Receiver operating characteristic analysis showed AACT was the best candidate for early NSCLC diagnosis of the four proteins, with 94.1% sensitivity in distinguishing early tumour Stage (IA+IB) from tumour-free samples (healthy and benign samples, HB). The GlcNAcylated AACT was further detected by lectin-based ELISA and has better advantage in clinical application than total AACT. The GlcNAcylated AACT can effectively differentiate Stage I from HB samples with an AUC of 0.908 and 90.9% sensitivity at a specificity of 86.2%. A combination of GlcNAcylated AACT and carcinoembryonic antigen (CEA) was able to effectively differing Stage I from HB samples (AUC=0.914), which significantly improve the specificity of CEA. The combination application also has the better clinical diagnostic efficacy in distinguishing cancer (NSCLC) from HB samples than CEA or GlcNAcylated AACT used alone, and yielded an AUC of 0.817 with 93.1% specificity.

Conclusions:

Our findings suggest that the GlcNAcylated AACT will be a promising clinical biomarker in diagnosis of early NSCLC.     

Quantitative proteomics analysis of differentially expressed proteins in activated B-cell-like diffuse large B-cell lymphoma using quantitative proteomics

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with unclear pathogenesis. DLBCL accounts for 30%–35% of all non-Hodgkin lymphomas (NHLs) and is an aggressive subtype of mature B-cell neoplasm. At present, half of DLBCL cases can be cured, although one-third of patients experience recurrence after treatment and enter advanced tumor stage. This study aimed to investigate the differentially expressed proteins in activated B-cell-like-DLBCL (ABC-DLBCL) through quantitative proteomics (iTRAQ). Seven ABC-DLBCL experimental samples and eight control samples (reactive hyperplasia of the lymph node) were obtained from fresh tissues. The exclusion criteria were expressed as follows: (1) patients with other lymphoid diseases; and (2) patients undergoing chemical treatment. A total of 5974 proteins were identified. P value < 0.05 and multiple expressions were more than 1.2-fold. A total of 131 upregulated and 204 downregulated differentially expressed proteins were identified. Gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis were performed. Protein–protein interaction (PPI) network analysis was conducted. The expression levels of HSP90AB1, GNA13, LAMB2, LAMA5, YWHAZ, and IKBKB were evaluated through PRM and TCGA to validate the accuracy of iTRAQ and liquid chromatography–tandem mass spectrometry results. Results of differential multiple and t-test showed differences in the expression levels of six target proteins between the control and experimental groups. To the best of our knowledge, the present study is the first to identify proteins associated with ABC-DLBCL using iTRAQ technology. Our results provide new insights into the pathogenesis of ABC-DLBCL. The combination of ABC-DLBCL-associated signaling pathway proteins and targeted therapy to reverse drug resistance is of great significance in improving the comprehensive treatment of lymphoma and reducing mortality of affected individuals. The feasibility of the present study is limited due to the number of samples, and future studies are required to determine the function of proteins in ABC-DLBCL development.     

Quantitative proteome profiling stratifies fibroepithelial lesions of the breast

Breast fibroepithelial lesions (FELs) include heterogeneous pathological tumors, involving indolent fibroadenoma (FAD) to potentially aggressive phyllodes tumors (PTs). The current grading system remains unreliable in differentiating these tumors due to histological heterogeneity and lack of appropriate markers to monitor the sudden and unpredictable malignant transformation of PTs. Thus, there exists an imminent need for a marker-based diagnostic approach to augment the conventional histological platform that could lead to accurate diagnosis and distinction of FELs. The high- throughput quantitative proteomic analysis suggested that FAD and PTs form distinct clusters away from borderline and malignant though there exist marked differences between them. Interestingly, over-expression of extracellular matrices (ECM) related proteins and epithelial-mesenchymal transition (EMT) markers in borderline PTs led us to hypothesize a model of deposition and degradation leading to ECM remodeling and EMT acquisition triggering its malignant transformation. We also identified three candidate biomarkers such as MUCL1, HTRA1, and VEGDF uniquely expressed in FAD, borderline, and malignant PTs, respectively, which were further validated using immunohistochemistry. The present work shed light on a brief mechanistic framework of PTs aggressive nature and present potential biomarkers to differentiate overlapping FELs that would be of practical utility in augmenting existing diagnosis and disease management for this rare tumor.     

绝对和相对定量的等量异位标签技术(iTRAQ)在中医药领域的应用

iTRAQ技术是采用同位素标记蛋白质中的多肽差异蛋白定量技术,能同时分析四或八个蛋白质样本。相对于传统的凝胶电泳蛋白组学技术和其它同位素标签技术有标本来源广、重复性高、检测面广、操作步骤简便等优势,为中药筛选、鉴定、开发和中医证型及临床研究提供了新的研究方法。文章从iTRAQ技术原理、较传统蛋白组学鉴别及定量研究的优势,并试综述其在中医药研究中的应用。     

MicroRNA-196a/-196b promote cell metastasis via negative regulation of radixin in human gastric cancer

MicroRNAs (miRNAs) play an important role to contribute carcinogenesis. The aim of the current study was to identify useful biomarkers from miRNAs. Differential miRNA profiles were analyzed using the miRNA qRT-PCR-based assay. Two of the most upregulated miRNAs were selected and validated. The miR-196a/-196b levels were significantly increased in gastric cancer (GC) tissues (n = 109). Overexpression of miR-196a/-196b was significantly associated with tumor progression and poorer 5-year survival outcomes. Overexpression of miR-196a/-196b enhances GC cell migration and invasion. Further, radixin was identified as a target gene of miR-196a/-196b. Elevated miR-196a/-196b expression in GC cells led to reduced radixin protein levels and vice versa. Notably, an inverse correlation between miR-196a/-196b and radixin mRNA and protein expression was observed in GC tissues with in situ hybridization and immunohistochemistry analyses. Together, miR-196a/-196b inhibitory oligonucleotides or overexpression of the radixin may thus have therapeutic potential in suppressing GC metastasis.     

应用iTRAQ标记结合2D LC-MS/MS分析慢性苯中毒患者血清蛋白质组

目的 比较慢性苯中毒患者与健康人的血清蛋白质组,筛选差异表达蛋白质,为阐明其发病机制和寻找生物标志物提供靶标.方法 免疫层析柱去除14种高丰度血清蛋白质后,应用8标iTRAQ技术结合2D LC-MS/MS分析并鉴定慢性苯中毒病例与健康人血清的差异表达蛋白质.结果 对9例慢性苯中毒患者和9名健康人的血清蛋白样品的iTRAQ标记,分析并成功鉴定159种蛋白质,其中差异表达有统计学意义(P＜0.05)的蛋白质有纤维蛋白溶酶原(PLG)、载脂蛋白B100( APOB100)、血小板碱性蛋白(PBP).这些差异表达蛋白质具有结合、催化、酶调节和转运活性等分子功能,参与的生物过程包括免疫、代谢、应激、转运和凋亡等.结论 PLG、APOB100和PBP可能作为慢性苯中毒发病机制研究的候选靶标.     

iTRAQ技术检测鼻息肉和正常鼻黏膜组织间差异表达蛋白质

目的 通过分析鼻息肉蛋白质表达谱的变化探索鼻息肉的发病机制。 方法 采集正常成年人中鼻道鼻腔黏膜以及鼻息肉患者的息肉组织(各4例),用同位素标记相对和绝对定量(iTRAQ)技术分析标本中的蛋白表达谱,筛选差异表达蛋白质。 结果 共筛选出差异表达蛋白311个。上调蛋白88个,主要包括CLC、PLEKHA7等,其中上调2倍以上的蛋白18个;下调蛋白223个,主要包括S100A1等,其中下调50%以上的蛋白65个。 结论 鼻息肉组织蛋白表达谱与正常中鼻道鼻腔黏膜相比有较大差异,差异蛋白可能在鼻息肉的发生发展过程中发挥重要作用,其中CLC可能参与鼻黏膜的炎症及免疫反应,PLEKHA7与鼻腔黏膜损伤修复有关。     

Selenium positively affects the proteome of 3 × Tg‐AD mice cortex by altering the expression of various key proteins: unveiling the mechanistic role of selenium in AD prevention

Selenium (Se) deficiency is believed to be involved in pathogenesis of Alzheimer's disease (AD) due to failure of antioxidant system. Its supplementation may restore the antioxidant system and compensate the impairments caused by AD. Present study reveals the effect of Se on the proteomic changes in cortex within triple transgenic male AD mice (3 × Tg‐AD) after 4 months sodium selenate supplementation. Using iTRAQ comparative proteomics approach, 142 proteins found significant alterations with 96 down‐regulated and 46 up‐regulated proteins in the cortices of AD mice in comparison with the wild non‐transgenic type mice. On treatment with sodium selenate, 41 proteins showed reverse expression, that is, thirty three proteins were down‐regulated in AD mice but up‐regulated in selenate treated AD mice while eight up‐regulated proteins in AD mice showed lower expression in selenate treated mice. OmicsBean bioinformatics analysis revealed that Se positively affected the proteins vital in biological process, structural cores, and molecular functions, which include metabolic proteins, structural proteins, signaling molecules, oxidative stress balancers, and proteosomal degradation proteins. Results of mass spectrometry (MS) were further confirmed by Western blot analysis of five important proteins, prompting the authenticity of the MS results. This paper fills the protein‐based molecular gap between AD and Se‐treatment, and it provides a full view of Se in reversing the change of cortical protein levels during AD formation.     

基于iTRAQ技术的慢性心力衰竭气虚血瘀证及气阴两虚证组差异蛋白质组学研究

目的:寻找慢性心力衰竭(CHF)(Chronic Heart Failure,CHF)气虚血瘀证及气阴两虚证的相关蛋白质组学特征,探讨证候发生发展机制的可能生物学通路。方法:研究设计分为CHF气虚血瘀证、气阴两虚证、健康对照组,利用i TRAQ标记定量结合串联质谱技术分析CHF气虚血瘀证及气阴两虚证的相关表达差异蛋白质,为蛋白质组学层面的疾病与证候生物学基础提供实验依据,可以在一定层面上揭示证的内涵。结果:与健康对照组比较,在CHF气虚血瘀证组表达差异蛋白质有16个,其中载脂蛋白E、半乳糖凝集素-3结合蛋白等11个蛋白质表达上调,维生素D结合蛋白、胰蛋白酶抑制剂等5个蛋白质表达下调;与健康对照组比较,在CHF气阴两虚证组表达差异的蛋白质有15个,其中补体9、间-α-胰蛋白酶抑制剂家族重链相关蛋白等10个蛋白质表达上调,前血清淀粉样蛋白P、维生素D结合蛋白等5个蛋白质表达下调。结论:蛋白质组检测结果能够反映CHF气虚血瘀证及气阴两虚证的生物学基础,从而能客观的评价证候特征,为病证结合的研究开拓了新的视野。