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Proteomics Experiments (proteomic + experiment)
Selected AbstractsProteomics of brain extracellular fluid (ECF) and cerebrospinal fluid (CSF),MASS SPECTROMETRY REVIEWS, Issue 1 2010Martin H. Maurer Abstract Mass spectrometry has become the gold standard for the identification of proteins in proteomics. In this review, I will discuss the available literature on proteomic experiments that analyze human cerebrospinal fluid (CSF) and brain extracellular fluid (ECF), mostly obtained by cerebral microdialysis. Both materials are of high diagnostic value in clinical neurology, for example, in cerebrovascular disorders like stroke, neurodegenerative diseases like Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis (ALS), traumatic brain injury and cerebral infectious and inflammatory disease, such as multiple sclerosis. Moreover, there are standard procedures for sampling. In a number of studies in recent years, biomarkers have been proposed in CSF and ECF for improved diagnosis or to control therapy, based on proteomics and mass spectrometry. I will also discuss the needs for a transition of research-based experimental screening with mass spectrometry to fast and reliable diagnostic instrumentation for clinical use. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 29:17,28, 2010 [source] MassSieve: Panning MS/MS peptide data for proteinsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2010Douglas J. Slotta Abstract We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments. [source] Experimental and computational tools useful for (re)construction of dynamic kinase,substrate networksPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2009Chris Soon Heng Tan Abstract The explosion of site- and context-specific in vivo phosphorylation events presents a potentially rich source of biological knowledge and calls for novel data analysis and modeling paradigms. Perhaps the most immediate challenge is delineating detected phosphorylation sites to their effector kinases. This is important for (re)constructing transient kinase,substrate interaction networks that are essential for mechanistic understanding of cellular behaviors and therapeutic intervention, but has largely eluded high-throughput protein-interaction studies due to their transient nature and strong dependencies on cellular context. Here, we surveyed some of the computational approaches developed to dissect phosphorylation data detected in systematic proteomic experiments and reviewed some experimental and computational approaches used to map phosphorylation sites to their effector kinases in efforts aimed at reconstructing biological signaling networks. [source] Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantificationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2009Jennifer J. Hill Dr. Abstract Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N -linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to ,2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine,protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. [source] Computational analysis of unassigned high-quality MS/MS spectra in proteomic data setsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2010Kang Ning Abstract In a typical shotgun proteomics experiment, a significant number of high-quality MS/MS spectra remain "unassigned." The main focus of this work is to improve our understanding of various sources of unassigned high-quality spectra. To achieve this, we designed an iterative computational approach for more efficient interrogation of MS/MS data. The method involves multiple stages of database searching with different search parameters, spectral library searching, blind searching for modified peptides, and genomic database searching. The method is applied to a large publicly available shotgun proteomic data set. [source] Semi-automatic tool to describe, store and compare proteomics experiments based on MIAPE compliant reportsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2010Salvador Martínez-Bartolomé Abstract The Human Proteome Organization's Proteomics Standards Initiative aims to develop new standards for data representation and exchange. The Proteomics Standards Initiative has defined the Minimum Information About a Proteomics Experiment (MIAPE) guidelines that specify the information that should be reported with a published experiment. With the aim of promoting the implementation of standard reporting guidelines, we have developed a web tool that helps to generate and store MIAPE compliant reports describing gel electrophoresis and MS-based experiments. The tool can be used in the reviewing phase of the proteomics publication process and can facilitate data interpretation through the comparison of related studies. [source] ICPLQuant , A software for non-isobaric isotopic labeling proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2010Achim Brunner Abstract The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope-coded protein label (ICPL)-labeled peptides on the MS level during LC-MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time-consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS-identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker. [source] Incorporating high-throughput proteomics experiments into structural biology pipelines: Identification of the low-hanging fruitsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2008Roland A. Pache Abstract The last years have seen the emergence of many large-scale proteomics initiatives that have identified thousands of new protein interactions and macromolecular assemblies. However, unfortunately, only a few among the discovered complexes meet the high-quality standards required to be promptly used in structural studies. This has thus created an increasing gap between the number of known protein interactions and complexes and those for which a high-resolution 3-D structure is available. Here, we present and validate a computational strategy to distinguish those complexes found in high-throughput affinity purification experiments that will stand the best chances to successfully express, purify and crystallize with little further intervention. Our method suggests that there are some 50 complexes recently discovered in yeast that could readily enter the structural biology pipelines. [source] A workflow to increase the detection rate of proteins from unsequenced organisms in high-throughput proteomics experimentsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2007Jonas Grossmann Abstract We present and evaluate a strategy for the mass spectrometric identification of proteins from organisms for which no genome sequence information is available that incorporates cross-species information from sequenced organisms. The presented method combines spectrum quality scoring, de novo sequencing and error tolerant BLAST searches and is designed to decrease input data complexity. Spectral quality scoring reduces the number of investigated mass spectra without a loss of information. Stringent quality-based selection and the combination of different de novo sequencing methods substantially increase the catalog of significant peptide alignments. The de novo sequences passing a reliability filter are subsequently submitted to error tolerant BLAST searches and MS-BLAST hits are validated by a sampling technique. With the described workflow, we identified up to 20% more groups of homologous proteins in proteome analyses with organisms whose genome is not sequenced than by state-of-the-art database searches in an Arabidopsis thaliana database. We consider the novel data analysis workflow an excellent screening method to identify those proteins that evade detection in proteomics experiments as a result of database constraints. [source] Development and validation of a spectral library searching method for peptide identification from MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2007Henry Lam Abstract A notable inefficiency of shotgun proteomics experiments is the repeated rediscovery of the same identifiable peptides by sequence database searching methods, which often are time-consuming and error-prone. A more precise and efficient method, in which previously observed and identified peptide MS/MS spectra are catalogued and condensed into searchable spectral libraries to allow new identifications by spectral matching, is seen as a promising alternative. To that end, an open-source, functionally complete, high-throughput and readily extensible MS/MS spectral searching tool, SpectraST, was developed. A high-quality spectral library was constructed by combining the high-confidence identifications of millions of spectra taken from various data repositories and searched using four sequence search engines. The resulting library consists of over 30,000 spectra for Saccharomyces cerevisiae. Using this library, SpectraST vastly outperforms the sequence search engine SEQUEST in terms of speed and the ability to discriminate good and bad hits. A unique advantage of SpectraST is its full integration into the popular Trans Proteomic Pipeline suite of software, which facilitates user adoption and provides important functionalities such as peptide and protein probability assignment, quantification, and data visualization. This method of spectral library searching is especially suited for targeted proteomics applications, offering superior performance to traditional sequence searching. [source] Automated reporting from gel-based proteomics experiments using the open source Proteios database applicationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2007Fredrik Levander Dr. Abstract The assembly of data from different parts of proteomics workflow is often a major bottleneck in proteomics. Furthermore, there is an increasing demand for the publication of details about protein identifications due to the problems with false-positive and false-negative identifications. In this report, we describe how the open-source Proteios software has been expanded to automate the assembly of the different parts of a gel-based proteomics workflow. In Proteios it is possible to generate protein identification reports that contain all the information currently required by proteomics journals. It is also possible for the user to specify maximum allowed false positive ratios, and reports are automatically generated with the corresponding score cut-offs calculated. When protein identification is conducted using multiple search engines, the score thresholds that correlate to the predetermined error rate are also explicitly calculated for proteins that appear on the result lists of more than one search engine. [source] Proteomic and transcriptomic analysis of rice mature seed-derived callus differentiationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2007Lan Yin Abstract Callus differentiation is a key developmental process for rice regeneration from cells. To better understand this complex developmental process, we used a 2-D gel electrophoresis approach to explore the temporal patterns of protein expression at the early stages during rice callus differentiation. This global analysis detected 60 known proteins out of 79 gel spots identified by MS/MS, of which many had been shown to play a role in plant development. Two new proteins were revealed to be associated with the callus differentiation and have been confirmed by Western blot analysis. The results of proteomics experiments were further verified at the mRNA level using microarray and real-time PCR. Comparison of the differentially expressed protein levels with their corresponding mRNA levels at the two callus early differentiation stages showed a good correlation between them, indicating that a substantial proportion of protein changes is a consequence of changed mRNA levels, rather than post-transcriptional effects during callus differentiation, though microarray revealed more expression changes on RNA levels. These findings may contribute to further understanding of the mechanisms that lead to callus differentiation of rice and other plants as well. [source] Printing of protein microarrays via a capillary-free fluid jetting mechanismPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2005J. A. Barron Abstract Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30,µm and ,500,fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer. [source] | ||||||||||