State of the art of 2D DIGE Arentz, Georgia; Weiland, Florian; Oehler, Martin K. ...
Proteomics. Clinical applications,
April 2015, Letnik:
9, Številka:
3-4
Journal Article
Recenzirano
Difference gel electrophoresis enables the accurate quantification of changes in the proteome including combinations of PTMs and protein isoform expression. Here, we review recent advances in study ...design, image acquisition, and statistical analysis. We also compare DIGE to established and emerging mass spectrometric analysis technologies. Despite these recent advances in MS and the still unsolved limitations of 2DE to map hydrophobic, high molecular weight proteins with extreme pIs, DIGE remains the most comprehensive top‐down method to study changes in abundance of intact proteins.
Serous endometrial cancer (SEC) and high grade serous ovarian cancer (HGSOC) are aggressive gynecological malignancies with high rates of metastasis and poor prognosis. Endometrial intraepithelial ...carcinoma (EIC), the precursor for SEC, and serous tubal intraepithelial carcinoma (STIC), believed to be the precursor lesion for HGSOC, can also be associated with intraabdominal spread. To provide insight into the etiology of these precancerous lesions and to explore the potential molecular mechanisms underlying their metastatic behavior, we performed a proteomic mass spectrometry analysis in a patient with synchronous EIC and STIC. Through histological and molecular identification of precancerous lesions followed by laser capture microdissection, we were able to identify over 450 proteins within the precancerous lesions and adjacent healthy tissue. The proteomic analysis of STIC and EIC showed remarkable overlap in the proteomic patterns, reflecting early neoplastic changes in proliferation, loss of polarity and attachment. Our proteomic analysis showed that both EIC and STIC, despite being regarded as premalignant lesions, have metastatic potential, which correlates with the common presentation of invasive serous gynecological malignancies at advanced stage.
Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% ...of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography-mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan-Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785.
Tumor biomarkers to more accurately predict a patient's response to a given therapy are much needed in oncology practice. For metastatic colorectal cancer the anti‐vascular endothelial growth factor ...(VEGF) monoclonal antibody bevacizumab is now commonly included in first‐line therapy regimens and has led to modest but significant improvements in patient outcomes compared with chemotherapy. Given the modest gains there is a pressing need for predictive biomarkers to better identify patients who would benefit from this targeted therapy. We used a multiplex protein assay to determine the tumor expression levels of the proangiogenic proteins IL‐6, IL‐8, bFGF, PDGF‐BB and VEGF‐A in formalin‐fixed paraffin‐embedded tumors from the MAX clinical trial patients with available tissue samples. Patients were dichotomized into “low” vs. “high” expression subgroups based on median baseline levels to correlate with objective response rate (ORR), progression‐free survival (PFS) and overall survival (OS). “Low” tumor VEGF‐A level was predictive of better ORR for bevacizumab ORR (low) 53% vs. (high) 19%, interaction p = 0.03 but not for PFS hazard ratio, HR (low) 0.73 vs. (high) 0.62, interaction p = 0.68 in the comparison of capecitabine (C) versus C and bevacizumab (CB) and CB plus mitomycin (M). When analyzed as a dichotomized variable, “high” VEGF‐A was prognostic for shorter PFS (unadjusted HR 1.34, p = 0.06; adjusted HR 1.55, p = 0.008). The other four proteins were neither predictive of bevacizumab benefits nor prognostic for ORR, PFS or OS. “Low” tumor VEGF‐A was associated with longer PFS after adjustment for other baseline factors. Proangiogenic proteins were not predictive of benefit with bevacizumab for PFS.
What's new?
There is a pressing need for predictive biomarkers to better identify metastatic colorectal cancer patients who would benefit from anti‐VEGF monoclonal antibody bevacizumab therapy. This study is the first to measure the expression levels of a panel of angiogenic proteins from FFPE tumors and to also use a multiplex assay platform‐‐an advantage given the limited amount of tissue available from clinical trials. Low tumor VEGF‐A was associated with significantly longer progression free survival after adjustment for other baseline factors. However neither VEGF‐A, nor the other angiogenic proteins IL‐6, IL‐8, bFGF or PDGF‐BB, were predictive of outcome for bevacizumab therapy.
The molecular analysis of small or rare patient tissue samples is challenging and often limited by available technologies and resources, such as reliable antibodies against a protein of interest. ...Although targeted approaches provide some insight, here, we describe the workflow of two complementary mass spectrometry approaches, which provide a more comprehensive and non-biased analysis of the molecular features of the tissue of interest. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) generates spatial intensity maps of molecular features, which can be easily correlated with histology. Additionally, liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify and quantify proteins of interest from a consecutive section of the same tissue. Here, we present data from concurrent precancerous lesions from the endometrium and fallopian tube of a single patient. Using this complementary approach, we monitored the abundance of hundreds of proteins within the precancerous and neighboring healthy regions. The method described here represents a useful tool to maximize the number of molecular data acquired from small sample sizes or even from a single case. Our initial data are indicative of a migratory phenotype in these lesions and warrant further research into their malignant capabilities.
Abstract Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic ...approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren’s syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.
Vulvar cancer is the fourth most common gynecological cancer worldwide. However, limited studies have been completed on the molecular characterization of vulvar squamous cell carcinoma resulting in a ...poor understanding of the disease initiation and progression. Analysis and early detection of the precursor lesion of HPV-independent vulvar squamous cell carcinoma (VSCC), differentiated vulvar intraepithelial neoplasia (dVIN), is of great importance given dVIN lesions have a high level of malignant potential. Here we present an examination of adjacent normal vulvar epithelium, dVIN, and VSCC from six patients by peptide Matrix-assisted laser desorption/ionization Mass Spectrometry Imaging (MALDI-MSI). The results reveal the differential expression of multiple peptides from the protein cytokeratin 5 (CK5) across the three vulvar tissue types. The difference observed in the relative abundance of CK5 by MALDI-MSI between the healthy epithelium, dVIN, and VSCC was further analyzed by immunohistochemistry (IHC) in tissue from eight VSCC patients. A decrease in CK5 immunostaining was observed in the VSCC compared to the healthy epithelium and dVIN. These results provide an insight into the molecular fingerprint of the vulvar intraepithelial neoplasia that appears to be more closely related to the healthy epithelium than the VSCC.
Retrospective proteomic studies, including those which aim to elucidate the molecular mechanisms driving cancer, require the assembly and characterization of substantial patient tissue cohorts. The ...difficulty of maintaining and accessing native tissue archives has prompted the development of methods to access archives of formalin-fixed tissue. Formalin-fixed tissue archives, complete with patient meta data, have accumulated for decades, presenting an invaluable resource for these retrospective studies. This review presents the current knowledge concerning formalin-fixed tissue, with descriptions of the mechanisms of formalin fixation, protein extraction, top-down proteomics, bottom-up proteomics, quantitative proteomics, phospho- and glycoproteomics as well as imaging mass spectrometry. Particular attention has been given to the inclusion of proteomic investigations of archived tumour tissue. This article is part of a Special Issue entitled: Medical Proteomics.
•Review on current knowledge for use of formalin-fixed tissue in proteomics•The following topics are discussed:•Fixation, protein extraction methods•Top down proteomics, bottom-up proteomics and quantitative proteomics•Specialized proteomics, including phospho, glyco and MALDI imaging
Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to ...greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker.
Protein from laser microdissected tumor and normal mucosa was analysed by two dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry to determine differentially over expressed tumor proteins. Tumor over-expression of one such protein, desmin, was quantified using immunofluorescence staining in a larger cohort. Dual staining for desmin and vimentin, or desmin and von Willebrand factor, was performed to determine the cell type of interest.
Desmin expression was significantly increased between stage I and III tumors, (P < 0.0001), and stage II and III tumors, (P < 0.0001). Strong focal desmin expression was found in stroma directly adjacent to carcinomatous glands and microvessels. These cells showed co-localisation of desmin and vimentin in close association with cells expressing VWF, indicating they were pericytes. Significantly higher levels of desmin-positive pericytes were observed in late stage tumors, consistent with increased angiogenesis.
Pericyte coverage of vasculature is a marker of vessel maturation, hence desmin expression may have use as a marker for microvessel maturation. Clinical trials will be needed to determine its use in identifying tumors that will be less responsive to anti-angiogenic therapy.
infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. ...Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen 2-phenyl-1,2-benzoselenazol-3(2H)-one is an inhibitor of
TrxR (
= 0.22 μM) and inhibits growth of
spp., with
MIC values of 16 to 64 µg/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of
TrxR and use
modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of
growth.
PDF
