Differential proteomic analysis of the reactivated p53 via Nutlin-3a, in 3 different types of human lymphomas

Purpose: The identification and quantification of protein expression levels of nutlin-3A-induced p53 stabilization and activation in human lymphoma. Methods: The Isotope Coded Protein Label (ICPL) technique was followed by nano-Liquid Chromatography coupled on-line with Mass Spectrometry (nLC-MS/MS). Results: Reliable identification & differential quantitative determination of human lymphoma proteome profile, revealing alterations in the HSPs relative expression levels

Datasets for transcriptomics, q-proteomics and phenotype microarrays of polyphosphate metabolism mutants from Escherichia coli

Indexación: Scopus.Author acknowledges Fondecyt Grants 1120209, 1121170 and Anillo ACT-1107Here, we provide the dataset associated with our research article on the polyphosphate metabolism entitled, “Multi-level evaluation of Escherichia coli polyphosphate related mutants using global transcriptomic, proteomic and phenomic analyses”. By integrating different omics levels (transcriptome, proteome and phenome), we were able to study Escherichia coli polyphosphate mutant strains (Δppk1, Δppx, and Δppk1-ppx). We have compiled here all datasets from DNA microarrys, q-proteomic (Isotope-Coded Protein Labeling, ICPL) and phenomic (Phenotype microarray) raw data we have obtained in all polyP metabolism mutants.http://www.sciencedirect.com/science/article/pii/S2352340917300860?via%3Dihu

Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients’ informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort

ICPL_ESIQuant – a Powerful Freeware Tool for Handling Proteomics LCESI- MS2 Experiments

Among the MS-based quantitative methods using stable isotope labelling, the Isotope-Coded Protein Label (ICPL) technique has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. The ICPL_ESIQuant 3.0 software package is one of the key components of the ICPL-ESI workflow, covering data processing steps like LC-MS feature detection, ICPL doublet/triplet/quadruplet quantification as well as a merging step of LC-MS features and Mascot search results. As unique features, the software performs isotope pattern overlap corrections and utilizes additional chemical knowledge, e.g. the physico-chemical properties of the ICPL labels, to discard false positive isotope pattern, which significantly improves the quality of the final peptide and protein results. ICPL_ESIQuant is the first freeware tool on the market, which supports both the shotgun proteomics strategy using Data Dependent Acquisition (DDA) and the directed proteomics strategy using mass inclusion lists for precursor ion selection. ICPL_ESIQuant 3.0 (32 and 64 bit versions) can be downloaded from https://sourceforge.net/projects/icplquant/ files

Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N -nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55852/1/5758_ftp.pd

Integrative analysis of the colorectal cancer proteome : potential clinical impact

Peer reviewedPostprin

Proteomic Analysis Suggests a Role for HtrA Serine Peptidase 1 in Immune Regulation and Thereby in Age-Related Macular Degeneration

Purpose. Single-Nucleotide Polymorphisms on chromosome 10 at the region 10q26 that harbors the three genes, PLEKHA1, ARMS2 and HtrA1, show strong association with the pathogenesis of age-related macular degeneration (AMD). In the last decade, evidence was mounted that mutations in the HtrA1 gene are a major causative factor of this disorder in Caucasian populations. The current study was undertaken in order to enhance the understanding the role of this HtrA1 in the pathogenesis of AMD. Methods. A HtrA1 gene trap mouse model was employed to compare the differences in protein expression between the gene trap mouse and a wild type mouse. ARPE19 and HeLa cells were transfected with a plasmid coding for triple-tagged, proteolytically inactive HtrA1. Next, a Co-IP assay was used to isolate HtrA1 interacting proteins. Selected candidates were validated with Western blot. Potential substrates of HtrA1 were tested by an in vitro digestion assay. Results. Our study indicated that HtrA1 as a secreted protein binds to various complement components, e.g. CFP, CFD, CFI, C1r, C1s, C2, C3 and C5 in the classical pathway, as well as to components of the lectin-mediated pathway and, in particular, to components of the alternative pathway. Several of these components have been implicated in the pathological process of age-related macular degeneration. Notably, a regulatory component of the innate immune activity, Ubiquitin-like protein ISG15 directly interacts with HtrA1 in our Co-IP. Furthermore, our in vitro digestion assay showed that Ubiquitin-like protein ISG15 is a substrate of HtrA1 protease. Conclusion. The aberrant activation of the complement system by HtrA1 found in our assays underlies the importance of the immune system in the pathogenesis of AMD. Our results suggest that HtrA1 may exert its biological function through the regulation of the immune system.ZIEL: Einzelnukleotid-Polymorphismen (englisch: Single-Nucleotide Polymorphisms, SNP) in dem chromosomalen Bereich 10q26, der die drei Gene PLEKHA1, ARMS2 und HtrA1 enthält, zeigen eine starke Assoziation mit der Pathogenese der Altersbedingten Makuladegeneration (AMD). In den letzten zehn Jahren haben sich zahlreiche Anzeichen dafür gehäuft, dass eine Hauptursache der Erkrankung in einer Mutation des HtrA1 Gens liegen könnte. Mit dieser Studie sollte daher die pathologische Rolle des Gens bzw. Proteins weiter aufgeklärt werden. METHODEN: Ein HtrA1 "gene trap" Mausmodell wurde verwendet, um die Proteinexpression zwischen der gene trap Maus und der Wildtyp Maus zu vergleichen. Des Weiteren wurden ARPE19 Zellen und HeLa Zellen mit einem HtrA1-Plasmid transfiziert, das einen dreifach Tag trägt und für proteolytisch inaktives HtrA1 kodiert. Mittels Co-Immunopräzipitation wurden dann die Proteine präzipitiert, die mit HtrA1 interagieren. Interessante Kandidaten wurden mit Western Blot überprüft. Potentielle HtrA1 Substrats, wurden mittels in vitro Verdau getestet. ERGEBNISSE: Die Ergebnisse dieser Studie zeigen, dass HtrA1 als ein sekretiertes Protein an verschiedene Komponenten des Komplementsystems binden kann, und zwar an die Komponenten CFP, CFD, CFI, C1r, C1s, C2, C3 und C5 aus dem klassischen Weg, sowie an Komponenten des Lektin-Weges und besonders an Komponenten des alternativen Weges. Einige dieser Proteine stehen im Zusammenhang mit der Pathogenese der Altersbedingten Makuladegeneration. Insbesondere konnten wir zeigen, dass das Ubiquitin-like protein ISG15 bei Co-Immunopräzipitation direkt mit HtrA1 interagiert. Bei einem in vitro Verdau war Ubiquitin-like protein ISG15 auch ein Substrat für HtrA1. AUSWERUNG: Die Aktivierung des Komplementsystems durch HtrA1, die wir unseren Assays gefunden haben, unterstützt die Vermutung, dass das Immunsystem eine entscheidende Rolle bei der Pathogenese von AMD spielt. Die Ergebnisse dieser Arbeit lassen vermuten, dass HtrA1 die Aktivität des Immunsystems reguliert

MASPECTRAS: a platform for management and analysis of proteomics LC-MS/MS data

<p>Abstract</p> <p>Background</p> <p>The advancements of proteomics technologies have led to a rapid increase in the number, size and rate at which datasets are generated. Managing and extracting valuable information from such datasets requires the use of data management platforms and computational approaches.</p> <p>Results</p> <p>We have developed the MAss SPECTRometry Analysis System (MASPECTRAS), a platform for management and analysis of proteomics LC-MS/MS data. MASPECTRAS is based on the Proteome Experimental Data Repository (PEDRo) relational database schema and follows the guidelines of the Proteomics Standards Initiative (PSI). Analysis modules include: 1) import and parsing of the results from the search engines SEQUEST, Mascot, Spectrum Mill, X! Tandem, and OMSSA; 2) peptide validation, 3) clustering of proteins based on Markov Clustering and multiple alignments; and 4) quantification using the Automated Statistical Analysis of Protein Abundance Ratios algorithm (ASAPRatio). The system provides customizable data retrieval and visualization tools, as well as export to PRoteomics IDEntifications public repository (PRIDE). MASPECTRAS is freely available at <url>http://genome.tugraz.at/maspectras</url></p> <p>Conclusion</p> <p>Given the unique features and the flexibility due to the use of standard software technology, our platform represents significant advance and could be of great interest to the proteomics community.</p

Поиск потенциальных биомаркеров хронических дерматозов с помощью протеомного анализа

The review covers the key achievements of proteome studies using mass spectrometry and two-dimensional electrophoresis methods in the field of dermatology Works studying the most prevalent chronic dermatoses such as psoriasis, atopic dermatitis, acne vulgaris and mycosis fungoidea are examined. Proteome analysis in dermatology is a promising technique today because it makes it possible to study molecular pathogenic mechanisms of skin chronic diseases in a greater detail. In addition, proteome technologies are aimed at searching for potential disease biomarkers and targets for drugs.В обзоре освещены основные достижения протеомных исследований с применением методов масс-спектрометрии и двумерного электрофореза в области дерматологии. Рассматриваются работы по изучению наиболее распространенных хронических дерматозов, таких как псориаз, атопический дерматит, вульгарные угри и грибовидный микоз. На сегодняшний день протеомный анализ в дерматологии является перспективным направлением, так как позволяет расширить знания о молекулярных механизмах патогенеза хронических заболеваний кожи. Кроме того, протеомные технологии направлены на поиск потенциальных биомаркеров заболеваний и мишеней для воздействия лекарственных средств