应用iTRAQ蛋白质组学方法筛选子痫前期患者胎盘组织差异蛋白

目的:应用同位素标记相对和绝对定量(i TRAQ)联合液相色谱-串联质谱(LC-MS/MS)技术筛选和鉴定正常组与子痫前期组胎盘组织差异蛋白,寻找潜在的子痫前期生物标记物。方法:收集2014年12月至2015年6月青岛大学附属医院产科收治的重度子痫前期患者30例(子痫前期组)和正常足月妊娠患者30例(正常组)。剖宫产术后取部分胎盘组织,提取总蛋白,对总蛋白进行变性、还原及酶解,i TRAQ标记后用质谱仪进行鉴定,得到表达差异蛋白。结果:(1)共鉴定到差异蛋白234个,子痫前期组较正常组表达丰度相差1.5倍以上(上调比值1.50或下调比值0.67),差异有统计学意义的蛋白质点共24个,其中14个蛋白点较正常组表达上调,10个点表达下调。结论:i TRAQ联合LC-MS/MS技术能有效筛选出子痫前期胎盘组织差异蛋白。     

Proteomic Analysis of Serum of Women with Elevated Ca-125 to Differentiate Malignant from Benign Ovarian Tumors

Clinically, elevated cancer antigen 125 (CA-125) in blood predicts tumor burden in a woman’s body, especiallyin the ovary, but cannot differentiate between malignant or benign. We here used intensive modern proteomicapproaches to identify predictive proteins in the serum of women with elevated CA-125 to differentiate malignantfrom benign ovarian tumors. We identified differentially expressed proteins in serum samples of ovarian cancer(OC) patients, benign ovarian tumor (BT) patients, and healthy control women using mass spectrometry-basedquantitative proteomics. Both the OC and BT patients had elevated CA-125. Quantitation was achieved usingisobaric tags for relative and absolute quantitation. We obtained 124 quantified differential serum proteins in OCcompared with BT. Two proteins, apolipoprotein A-4 (APOA4) and natural resistance-associated macrophage1, were verified using Western blotting. Proteome profiling applied to OC cases identified several differentialserum proteins in the serum of women with elevated CA-125. A novel protein, APOA4, has the potential to bea marker for malignant tumor differentiation in the serum of women with elevated CA-125.     

Proteomics analysis of differential protein expression identifies heat shock protein 47 as a predictive marker for lymph node metastasis in patients with colorectal cancer

The discovery of biomarkers to predict the potential for lymph node (LN) metastasis in patients with colorectal cancer (CRC) is essential for developing improved strategies for treating CRC. In the present study, they used isobaric tags for relative and absolute quantitation to conduct a proteomic analysis designed to identify novel biomarkers for predicting LN metastasis in patients with CRC. They identified 60 differentially expressed proteins specifically associated with LN metastasis in CRC patients and classified the molecular and functional characteristics of these proteins by bioinformatic approaches. A literature search led them to select heat shock protein 47 (HSP47) as the most suitable candidate biomarker for predicting LN metastasis. Validation analysis by immunohistochemistry showed that HSP47 expression in patients with CRC and the number of HSP47‐positive spindle cells in the tumor stroma were significantly higher compared with those in adjacent normal colonic mucosa, and the number of the latter cells increased with tumor progression. Further, the number of HSP47‐positive spindle cells in stroma was a more informative marker for identifying LN metastasis than HSP47expression. Multivariate analysis identified spindle cells that expressed elevated levels of HSP47 as an independent predictive biomarker for CRC with LN metastasis. Moreover, these cells served as an independent marker of disease‐free and overall survival of patients with CRC. Their data indicate that the number of HSP47‐positive spindle cells in the stroma of CRC may serve as a novel predictive biomarker of LN metastasis, early recurrence and poor prognosis.     

Biomarkers of chemotherapy resistance in breast cancer identified by proteomics: Current status

This review describes and discusses the advantages and limitations of proteomic approaches in the identification of biomarkers associated with chemotherapy resistance. Both gel-based (two-dimensional polyacrylamide gel electrophoresis) and gel-free (shotgun and quantitative) mass spectrometry approaches are discussed. Non-mass spectrometry approaches including antibody microarray platforms are described as complementary proteomic strategies. Methods for technical confirmation and clinical validation of putative biomarkers are presented. Use of this proteomic toolbox in the quest for biomarkers of chemotherapy resistance in breast cancer is reviewed. Technical aspects of sample selection, acquisition, storage and analysis are discussed and putative biomarkers identified through proteomic approaches are presented.     

SAA与脓毒症进程的相关性及其生物学意义

目的通过蛋白质组方法研究CLP模型小鼠,研究脓毒症进程相关的生物标志分子。 方法85只小鼠,分成模型组和假手术组,制作小鼠CLP脓毒症模型,观察脓毒症发生发展的自然过程,同时在关键时间点采用iTRAQ法检测和鉴定血清蛋白,确定与脓毒症进程密切相关的关键分子,并通过qPCR法观察不同组织的mRNA表达,判断其组织来源。 结果生存曲线显示,脓毒症小鼠发病12～32 h是死亡发生的关键时间,度过这一时间则进入自然康复期。同时发现一组蛋白在出现、高峰和恢复时间上与这一进程匹配。蛋白鉴定证实,这组蛋白包括SAA1和SAA2,两者表达水平相当,表达规律相同。检查不同组织SAA1 mRNA表达发现,肝脏是合成SAA1的主要场所,肝外组织也能合成少量SAA1。脓毒症组小鼠外周血出现单体形态的SAA1和SAA2蛋白。 结论SAA可能具有多种生物学功能,脓毒症外周血血清SAA是SAA1和SAA2的混合物,肝外组织也能够合成一定量的SAA,血清中的单体形态的SAA可以作为脓毒症的生物标志分子。     

iTRAQ技术在消化道肿瘤研究中的应用进展

近年来,同位素标记相对和绝对定量(Isobaric tags for relative and absolute quantification,iTRAQ)技术已成为蛋白质组学定量研究中一种强有力的工具,尤其在肿瘤相关领域备受关注,其突出优势为测定蛋白范围广泛、分析结果可靠、精确度高、重复性好等。大量文献也表明,iTRAQ技术在消化道肿瘤,如食管癌、胃癌、肝癌等肿瘤标志物筛查研究、肿瘤发生发展机制研究以及肿瘤治疗预后探索等方面得到广泛应用。在大规模定量生物学时代,iTRAQ技术在分子水平上深入了解消化道肿瘤的相关机制研究中越来越不可或缺。因此本文对iTRAQ技术在消化道肿瘤中的研究进展进行简要综述。     

Quantitative Proteomic Analysis of Peripheral Blood Mononuclear Cells in Ankylosing Spondylitis by iTRAQ

This study was designed to identify and quantify the different proteins expression levels in ankylosing spondylitis (AS) and to explore the pathogenesis of AS. We performed isobaric tags for relative and absolute quantitation (iTRAQ) coupled with multiple chromatographic fractionation and tandem mass spectrometry to detect the proteins profiling in peripheral blood mononuclear cells (PBMCs) from AS patients and healthy controls. Mascot software and the International Protein Index and the Gene Ontology (GO) database were used to conduct the bioinformatics analysis. The differentially expressed proteins were validated by enzyme‐linked immunosorbent assay (ELISA). A total of 1,232 proteins were identified by iTRAQ, of which 183 showed differential expression and 18 differentially expressed proteins were acute phase reactants. Upon mapping of the differentially expressed proteins to GO database, we found four differentially expressed proteins involved in the biological process of cell killing, including up‐regulated cathepsin G (CTSG), neutrophil defensin3 (DEFA3), protein tyrosine phosphatase receptor type C (PTPRC), and down‐regulated peroxiredoxin‐1(PRDX1),which were consistent with the verified results of ELISA. Our proteomic analyses suggested that the proteins involved in the biological process of cell killing might play an important role in the pathogenesis of AS.     

iTRAQ-based quantitative analysis of alveolar bone resorption in rats with experimental periodontitis

ObjectivePeriapical periodontitis results in alveolar bone resorption around the root apex. During the progression of inflammation, host cells release various inflammatory mediators and pro-inflammatory cytokines through immune responses. However, the pathological mechanisms associated with periapical bone destruction remain unclear. This study was objected to identify differentially regulated proteins in periapical periodontitis via a quantitative proteomics approach using isobaric tags for relative and absolute quantification (iTRAQ) labelling of peptides.MethodsA model of periapical periodontitis by sealing LPS into the pulp chambers of rats was established. iTRAQ was employed to screen differentially expressed proteins in alveolar bone between periapical lesions and healthy controls. These proteins were further analysed by bioinformatics. And four proteins were validated by western bolt.ResultsWe identified 4398 proteins, of which 7 were up-regulated and 151 were down-regulated in periapical periodontitis compared to normal tissue. Using bioinformatics tools such as GO and KEGG pathway analysis, we found that our proteomics strategy could identify and quantify differentially expressed proteins that were not described in previous studies examining periapical periodontitis; these proteins included hexokinase, legumain and members of the keratin family.ConclusionIn summary, our results represent potential biomarkers for the detection of periapical periodontitis and demonstrate that quantitative proteomics is a robust discovery tool for the identification of differentially regulated proteins in periapical periodontitis.