iTRAQ多重标记串联质谱技术在乳腺癌细胞侵袭患者HGF表达中的应用

目的探讨iTRAQ多重标记串联质谱技术应用于检测乳腺癌细胞侵袭患者肝细胞生长因子(HGF)表达差异的临床价值。方法选择该院2014年1月至2016年10月收治的35例乳腺癌患者与30例健康者为研究对象,通过iTRAQ标记、质谱检测、搜库以及Scqffold软件分析不同临床分期乳腺癌患者与健康者血清HGF表达差异,并用Western blot验证HGF的差异表达。结果该研究血清样品中共鉴定出蛋白237个,达到严格定量标准的蛋白89个,乳腺癌患者与健康者共筛选出包括HGF在内的差异表达蛋白17个;iTRAQ多重标记串联质谱图谱显示,乳腺癌患者血清HGF表达水平显著高于健康者,差异有统计学意义(P0.05);Western blot结果显示,不同临床分期HGF相对表达水平显著高于健康者,差异有统计学意义(P0.05)。结论iTRAQ多重标记串联质谱技术有助于发现乳腺癌患者癌细胞高表达HGF,对指导临床治疗乳腺癌具有重要意义。     

同位素标记相对和绝对定量技术在药物蛋白质组学研究中的应用

目的建立应用同位素标记相对和绝对定量(iTRAQ)技术进行药物蛋白质组学研究的技术平台。方法分别测定12例高血压患者在给予降压药氨氯地平治疗前和治疗后8周的收缩压和舒张压,并同时收集患者在降压药治疗前和治疗后8周的血清,应用蛋白质组学iTRAQ技术对治疗前后的血清蛋白质组进行定量分析。结果高血压患者口服降压药苯磺酸氨氯地平片8周后平均收缩压和舒张压均明显降低(P<0.01)。经iTRAQ和ProteinPilot软件分析,共鉴定出232个差异蛋白质,其中降压药物治疗后表达上调和下调超过1.5倍的蛋白质分别有12种和13种。上调的蛋白质主要参与应激、防御和免疫反应,而下调的蛋白质主要参与应激、防御、炎症和凝血反应。本研究结果提示,降压药氨氯地平治疗可以双向调节机体的应激和防御反应,可以增强机体的免疫力,抑制炎症反应和改善高血凝状态,从而有利于血压的降低。结论应用iTRAQ技术进行药物蛋白质组学研究可筛选获得多种与药物疗效相关的差异蛋白质,为药物作用靶点和分子机制等方面的研究提供了一个良好的技术平台。     

Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC‐derived exosomes on angiogenesis. Exosomes derived from the NPC C666‐1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666‐1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose‐dependent manner. Subsequently, an iTRAQ‐based quantitative proteomics was used to identify the differentially expressed proteins in C666‐1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up‐ and down‐regulated (≥ 1.5‐fold variations) in C666‐1 exosomes compared to the normal counterparts, respectively. As expected, pro‐angiogenic proteins including intercellular adhesion molecule‐1 (ICAM‐1) and CD44 variant isoform 5 (CD44v5) are among the up‐regulated proteins, whereas angio‐suppressive protein, thrombospondin‐1 (TSP‐1) was down‐regulated in C666‐1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM‐1 and TSP‐1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes‐induced angiogenesis, which could potentially be developed as therapeutic targets in future.     

基于iTRAQ技术筛选室间隔缺损患儿血清蛋白标志物

目的:应用同位素标记相对和绝对定量(iTRAQ)技术结合LC-MALDI-TOF/TOF方法比较室间隔缺损(VSD)患儿和健康对照组儿童血清中的差异表达蛋白质,筛选并鉴定潜在的VSD血清蛋白标志物。方法:收集VSD患儿和与之相匹配的健康儿童血清各30例,组内等量混合后利用多重免疫亲和层析柱(MARS)去除14种高丰度蛋白,iTRAQ试剂标记后的样本应用二维液相色谱分离,采用MALDI-TOF/TOF质谱鉴定及相对定量。结果:质谱鉴定出置信度>95%的蛋白质共166种,其中差异表达蛋白质21种,VSD组较健康对照组表达量上调≥1.5倍的蛋白质有13种,下调≤0.67倍的蛋白质有8种。结论:筛选出多种与VSD相关的差异表达蛋白质,为进一步验证VSD血清蛋白标志物奠定了基础。     

急性脊髓损伤模型大鼠蛋白质组学研究：iTRAQ技术的验证

背景：同位素标记相对和绝对定量（Isobaric tags for relative and absolute quantitation,iTRAQ）质谱分析技术依据串联质谱中信号离子表达质荷比峰值的不同来研究相关对应蛋白质的信息。目的：建立急性脊髓损大鼠模型,观察其脑脊液差异蛋白谱,从微观分子水平研究急性脊髓损伤后继发性损伤的机制及有效治疗方法。方法：建立SD大鼠急性脊髓损伤模型,取脑脊液应用iTRAQ技术鉴定SD大鼠急性脊髓损伤后脑脊液的差异蛋白质。结果与结论：共鉴定蛋白数722个,差异表达蛋白107个：下调的差异蛋白有63个,上调的差异蛋白数是44个。其中相关神经再生的差异蛋白19个：上调14个,下调5个;调节神经再生的差异蛋白7个。实验中检测到的多种差异蛋白及表达明显的神经再生因子可能作为急性脊髓损伤的生物标记物或可能作为临床管理监测急性脊髓损伤的损伤进程、靶向治疗及评估疗效的强有力证据。     

Simultaneous proteomic profiling of four different growth states of human fibroblasts, using amine-reactive isobaric tagging reagents and tandem mass spectrometry

In general, permanent growth arrest due to exhaustive cell replication can be induced prematurely by either stress or overexpression of selected oncogenes. In an attempt to examine key proteins involved in achieving premature senescence, and how they differ from those in serially passaged, replicatively exhausted cells, we used a novel proteomic profiling approach, isobaric tagging for relative and absolute quantitation (iTRAQ), to perform simultaneous four-way comparison of replicatively senescent fibroblasts, oxidatively stressed prematurely senescent fibroblasts, and their young replicating and quiescent counterparts. Two hundred and forty proteins were identified and quantified simultaneously; data analysis reveals: (1) groups of proteins whose expressions are uniformly either up- or down-regulated in all three growth arrest states; (2) signature proteins which may serve as candidate proteomic markers to differentiate the quiescent state from permanent growth arrest by either exhaustive replication or stress induction and (3) that while oxidative stress-induced, prematurely senescent fibroblasts morphologically resemble their replicatively exhausted counterparts, they exhibit different protein expression patterns. Results from simultaneous proteomic profiling were validated by Western blotting for selected proteins: collagen type I, HSP90 and vimentin. In conclusion, this report shows that iTRAQ proteomic profiling is a powerful technique for globally mapping protein signatures for different culture growth states.     

空间环境诱导屎肠球菌突变株LCT-EF18的蛋白组学分析

目的 对搭载神舟八号飞船的屎肠球菌进行突变株筛选,并鉴定和分析差异表达的蛋白质。方法 利用Biolog 生化反应板从搭载神舟八号飞船飞行后屎肠球菌中筛选突变株,提取突变株蛋白质并进行SDS-PAGE 电泳检测,用BCA 法测定总蛋白浓度后,采用同重同位素相对与绝对定量（isobaric tags for relative and absolute quantitation,iTRAQ） 方法分析空间环境诱变屎肠球菌突变株和地面对照株之间的差异表达蛋白,并进行统计、分析和注释。结果 通过Biolog 表型筛选获得空间环境诱导屎肠球菌突变株LCT-EF20,该菌株能利用肌酐和L- 苹果酸,而不能利用对羟基苯乙酸。差异蛋白组学分析发现了124 个蛋白质表达的改变,其中50 个蛋白表达上调,74 个蛋白表达下调。结论 太空环境可改变屎肠球菌的生物学特性,并影响大量蛋白质的表达,差异表达蛋白主要分布在代谢相关过程。     

Comparative physiological and proteomic analyses reveal the actions of melatonin in the reduction of oxidative stress in Bermuda grass (Cynodon dactylon (L). Pers.)

The fact of melatonin as an important antioxidant in animals led plant researchers to speculate that melatonin also acts in the similar manner in plants. Although melatonin has significant effects on alleviating stress‐triggered reactive oxygen species (ROS), the involvement of melatonin in direct oxidative stress and the underlying physiological and molecular mechanisms remain unclear in plants. In this study, we found that exogenous melatonin significantly alleviated hydrogen peroxide (H₂O₂)‐modulated plant growth, cell damage, and ROS accumulation in Bermuda grass. Additionally, 76 proteins significantly influenced by melatonin during mock or H₂O₂ treatment were identified by gel‐free proteomics using iTRAQ (isobaric tags for relative and absolute quantitation). Metabolic pathway analysis showed that several pathways were markedly enhanced by melatonin and H₂O₂ treatments, including polyamine metabolism, ribosome pathway, major carbohydrate metabolism, photosynthesis, redox, and amino acid metabolism. Taken together, this study provides more comprehensive insights into the physiological and molecular mechanisms of melatonin in Bermuda grass responses to direct oxidative stress. This may relate to the activation of antioxidants, modulation of metabolic pathways, and extensive proteome reprograming.     

Proteomic and bioinformatic analysis of condyloma acuminata: mild hyperthermia treatment reveals compromised HPV infectivity of keratinocytes via regulation of metabolism,differentiation and anti-viral responses

Background: Hyperthermia has proved successful in treating cutaneous human papillomavirus infectious diseases such as plantar wart and condyloma acuminata (CA). Moreover, this treatment provides improved therapeutic efficacy in these conditions as compared with conventional therapies.

Objectives: To investigate the global proteome changes in CA in response to hyperthermia and achieve a better understanding of the mechanisms of hyperthermia therapy against HPV-infectious diseases.

Methods: CA tissue was obtained from patients undergoing pathological examinations. Diagnosis was verified as based on results of both HE staining and HPV-DNA PCR assay. Hyperthermia was achieved with a 44?°C water bath. Differentially expressed proteins (DEPs) were identified by iTRAQ labeling, SCX chromatography and LC-MS/MS assay. Validation of proteomic results was performed using real-time qPCR and western blot, while bioinformatic analysis of DEPs was accomplished by R 3.4.1, STRING and Cytoscape softwares.

Results: In response to hyperthermia, a total of 102 DEPs were identified with 37 being upregulated and 65 downregulated. Among these DEPs, hyperthermia induced proteins involved with anti-viral processes such as OAS1, MX1, BANF1, CANX and AP1S1, whereas it inhibited proteins that participated in cellular metabolism, such as GALT, H6PD, EXOSC4 and EXOSC6; protein translation, such as RPS4Y1; as well as keratinocyte differentiation, such as KRT5, KRT27, KRT75, KRT76 and H2AFY2.

Conclusions: Hyperthermia inhibited enzymes and molecules responsible for metabolism modulation and keratinocyte differentiation in CA tissue, whereas it promoted factors involved in anti-viral responses. Such effects may, in part, contribute to the efficacy of local hyperthermia therapy against HPV infection.