Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “quantitative proteomics”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,369 records · Page 76Linked to original sources
PubMed2006

Improved immobilized metal affinity chromatography for large-scale phosphoproteomics applications.

Dysregulated protein phosphorylation is a primary culprit in multiple physiopathological states. Hence, although analysis of signaling cascades on a proteome-wide scale would provide significant insight into both normal and aberrant cellular function, such studies are simultaneously limited by sheer biological complexity and concentration dynamic range. In principle, immobilized metal affinity chromatography (IMAC) represents an ideal enrichment method for phosphoproteomics. However, anecdotal evidence suggests that this technique is not widely and successfully applied beyond analysis of simple standards, gel bands, and targeted protein immunoprecipitations. Here, we report significant improvements in IMAC-based methodology for enrichment of phosphopeptides from complex biological mixtures. Moreover, we provide detailed explanation for key variables that in our hands most influenced the outcome of these experiments. Our results indicate 5- to 10-fold improvement in recovery of singly- and multiply phosphorylated peptide standards in addition to significant improvement in the number of high-confidence phosphopeptide sequence assignments from global analysis of cellular lysate. In addition, we quantitatively track phosphopeptide recovery as a function of phosphorylation state, and provide guidance for impedance-matching IMAC column capacity with anticipated phosphopeptide content of complex mixtures. Finally, we demonstrate that our improved methodology provides for identification of phosphopeptide distributions that closely mimic physiological conditions.

Amino Acid Sequence↗
PubMed2002

Protein chips: from concept to practice.

A series of exciting reports over the past two years has established the usefulness of protein chips and made important advances in preparing protein arrays. However, several technical challenges must still be addressed to make these tools available to the wider community of researchers. Here, we discusses these challenges and survey recent opportunities for creating quantitative assays, preparing and immobilizing large numbers of proteins, using detection methods to analyze the results of chip-based experiments, and using informatics tools to interpret these results.

Biotechnology↗
PubMed2006

Phosphoproteomics in Arabidopsis: moving from empirical to predictive science.

Although protein phosphorylation is integral to the regulation of protein function in diverse biological responses, relatively little is currently known about the rules that govern phosphorylation in plants. This review will discuss how the data acquired by evolving phosphoproteomic methods are beginning to fill the gaps in our knowledge. In addition, ways are suggested in which new quantitative methods in conjunction with extrapolating from genomic data may provide a strategy to predict components of signalling networks that may be co-ordinately regulated.

Amino Acid Motifs↗
PubMed2005

Proteomic analysis of rat hippocampus after repeated psychosocial stress.

Since stress plays a role in the onset and physiopathology of psychiatric diseases, animal models of chronic stress may offer insights into pathways operating in mood disorders. The aim of this study was to identify the molecular changes induced in rat hippocampus by repeated exposure to psychosocial stress with a proteomic technique. In the social defeat model, the experimental animal was defeated by a dominant male eight times. Additional groups of rats were submitted to a single defeat or placed in an empty cage (controls). The open field test was carried out on parallel animal groups. The day after the last exposure, levels of hippocampal proteins were compared between groups after separation by 2-D gel electrophoresis and image analysis. Spots showing significantly altered levels were submitted to peptide fingerprinting mass spectrometry for protein identification. The intensity of 69 spots was significantly modified by repeated stress and 21 proteins were unambiguously identified, belonging to different cellular functions, including protein folding, signal transduction, synaptic plasticity, cytoskeleton regulation and energy metabolism. This work identified molecular changes in protein levels caused by exposure to repeated psychosocial stress. The pattern of changes induced by repeated stress was quantitatively and qualitatively different from that observed after a single exposure. Several changed proteins have already been associated with stress-related responses; some of them are here described for the first time in relation to stress.

Animals↗
PubMed2026

A Parallel Accumulation-Mobility Aligned Fragmentation Strategy Utilizing High-Resolution Ion Mobility for High-Performance Proteomics Analysis.

Here we present a novel data-independent acquisition (DIA) mass spectrometry (MS) operating mode termed parallel accumulation-mobility aligned fragmentation (PAMAF) that offers enhanced speed and sensitivity of ion fragmentation analysis for discovery workflows such as bottom-up proteomics. This mode of operation leverages high-resolution ion mobility (HRIM) separation capabilities of the structures for lossless ion manipulation technology to achieve HRIM-based precursor isolation in place of traditional quadrupole filtering approaches. PAMAF mode increases the number of features that can be identified per MS1/MS2 acquisition cycle by employing mobility-based time alignment to associate fragment ions with their corresponding precursor ions. By using a high-speed, lossless separation technique for precursor isolation instead of the comparatively slow and wasteful quadrupole filtering, ion losses are avoided while simultaneously increasing the rate at which precursor ions are sequentially fragmented and detected. In addition, by accumulating ions while the previous packet of ions is being analyzed, the PAMAF mode achieves ∼100% ion utilization efficiency. Benchmarking results of LC-PAMAF-MS analysis of a whole cell protein digest showed ∼6× more protein group identifications compared to a standard data-dependent acquisition analysis without HRIM on the same QTOF instrument, and >100 x improvement for low-load workflows. Quantitative evaluations demonstrated that PAMAF mode could quantify low abundance peptides, including those undetectable by data-dependent acquisition. In addition, since precursor isolation in PAMAF mode is size-based rather than m/z-based, coeluting isobars and isomers can be resolved prior to fragmentation, eliminating chimeric spectra that compromise identification accuracy. We also explored the benefits of combining HRIM and quadrupole isolation to achieve improved specificity termed DIA-PAMAF mode, which enabled the detection of over 8000 protein groups from a HeLa digest analysis. PAMAF mode brings a powerful new technique to the field of proteomics with the potential to improve the sensitivity and selectivity of mass spectrometry-based proteomics.

Proteomics↗
PubMed2003

Newly developed techniques to study and diagnose acute renal failure.

Progress in treating human acute renal failure (ARF) is dependent on developing techniques that allow for the rapid diagnosis, quantification of injury, further understanding of the pathophysiology, and the effects of therapy. Therefore, four techniques that will facilitate this progress are described and illustrated by four different investigative teams. Techniques to measure rapid changes in GFR are available for rapid diagnosis and quantification of ARF in humans. State-of-the-art magnetic resonance imaging (MRI) presently allows for enhanced resolution of regional renal blood flow and functional evaluations in patients. Furthermore, new probes and techniques for MRI that allow for identification and quantitation of inflammation, applicable to human ARF, are being developed and tested in animal models. Finally, two-photon microscopy will allow for four-dimensional cellular and subcellular studies in animal models of ARF providing rapid insights into pathophysiology and the therapeutic effects of a variety of promising agents. Further development and utilization of these techniques, especially in concert with genetic, proteomic, and molecular approaches, will allow for needed insights into the pathophysiology and therapy in human ARF.

Acute Kidney Injury↗
PubMed2003

[Studies on the simvastatin effect on the artery of atherosclerotic rabbit using proteomics approaches].

AIM: To evaluate the inhibitory effect of simvastatin via investigating the overall expression level of proteins in the artery of atherosclerotic rabbit. METHODS: Experimental model was established by feeding the rabbits a high fat diet (cholesterol 0.5 g.kg-1.d-1, lard 0.5 mL.kg-1.d-1) for 8 weeks. Then simvastatin (5 mg.kg-1) were fed for 4 weeks to the rabbits. The overall protein levels were measured using two-dimensional gel electrophoresis and a PDQUEST data processing. RESULTS: Twenty nine protein spots showed significant quantitative changes in comparison with the normal and the diseased rabbits. Furthermore, after the diseased rabbit having taken simvastatin, an obvious decay of symptom of fatty liver was observed, and the intensity of most spots has not been back-regulated. CONCLUSION: Simvastatin facilitates the metabolism of fat in the blood, but the lesion of the internal wall of the atherosclerotic artery cannot be restored.

Animals↗
PubMed2005

Analysis of leaf proteome after UV-B irradiation in maize lines differing in sensitivity.

UV-B radiation causes diverse morphological and physiological responses in plants, but the underlying mechanisms governing these integrated responses are unknown. In this study, we systematically surveyed responses of maize leaves to UV-B radiation using DIGE 2D gels and identified selected proteins by mass spectrometry and immunodetection analysis. To identify changes in protein accumulation in response to UV-B radiation, a line (b, pl W23) deficient in flavonoid sunscreen compounds and hence similar to commercial corn was used. In addition, its proteome in natural UV-B conditions was compared with that of two maize landraces from high altitudes (Cacahuacintle and Confite Puneño) that have improved UV-B tolerance. Protein patterns in adult maize leaves (Zea mays) were documented after growth for 21 days in sunlight depleted of UV-B radiation or growth in sunlight including an 8-h UV-B supplementation during 1 day in the field. We found that there is a very high correlation between previously documented mRNA accumulation assessed by microarray hybridization and quantitative real time reverse transcription-PCR and protein expression after UV-B irradiation in leaves of W23. Multiple isoforms were confirmed for some proteins; at least one protein, pyruvate, phosphate dikinase, is regulated post-translationally by phosphorylation by UV-B exposure. Proteins differentially regulated by UV-B radiation in W23 with higher levels under similar UV-B conditions in high altitude plants were also identified. These could be genetically fixed traits conferring UV-B tolerance and offer clues to specific adaptations to living in high ambient UV-B conditions.

Amino Acid Sequence↗
PubMed1999

Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level.

We have investigated how the programme of protein synthesis is altered in response to a loss of calcium homoeostasis in Cos-7 cells using a differential proteome mapping approach. Exposure of the cells to the calcium ionophore A23187 or thapsigargin, or alternatively, expression of a viral glycoprotein reported to deplete intracellular calcium stores, resulted in the up-regulated expression of a characteristic set of proteins. One of these is the translationally controlled tumour protein (TCTP), a cytoplasmic protein whose expression has not previously been linked to calcium perturbation. Quantitative Northern blot assay demonstrated that steady-state mRNA abundance of TCTP was also increased under these conditions. Clamping the cytosolic calcium concentration by the introduction of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) into cells did not affect the increase in steady-state levels of TCTP mRNA observed in response to ionophore. Therefore depletion of endoplasmic reticulum (ER) calcium, but not elevation of the cytosolic calcium concentration, was responsible for increased transcription of the TCTP gene. However, the presence of BAPTA significantly attenuated the ionophore-mediated increase in levels of the protein. Moreover, the level of TCTP in ionophore-treated cells increased in advance of a detectable increase in the corresponding mRNA abundance. These results indicate that expression of TCTP is regulated at two distinct levels in response to the concentration of calcium in different cellular compartments. Whereas depletion of the ER store causes an increase in TCTP mRNA abundance, increased cytosolic calcium concentrations regulate gene expression at the post-transcriptional level.

Animals↗
PubMed2004

High-field asymmetric waveform ion mobility spectrometry: a new tool for mass spectrometry.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a new technology for ion separation at atmospheric pressure. This review introduces the reader to FAIMS, covering topics ranging from the fundamentals and extraction of physical parameters from the raw data, to applications of FAIMS extending from homeland security to environmental analysis to proteomics. The investigation of FAIMS as an ion pre-processing tool for mass spectrometry is in its infancy, but reports in the literature illustrate that FAIMS separates isobaric ions including diastereoisomers, separates isotopes, reduces background ions by isolating ions of interest, and simplifies spectra of complex mixtures by dividing the mixture into a series of simpler subsets of ions. Applications ranging from quantitative analysis of inorganic and organometallic compounds, to studies of the conformers of intact proteins, have been reported. This review is a launching point for further exploration of FAIMS.

Mass Spectrometry↗
PubMed2006

Proteomic identification of altered proteins in skeletal muscle during chronic potassium depletion: Implications for hypokalemic myopathy.

Prolonged potassium depletion is a well-known cause of myopathy. The pathophysiology of hypokalemic myopathy, however, remains unclear. We performed a gel-based, differential proteomics study to define altered proteins in skeletal muscles during chronic potassium depletion. BALB/c mice were fed with normal chow (0.36% K+) or K+-depleted (KD) diet (<0.001% K+) for 8 weeks (n = 5 in each group). Left gastrocnemius muscles were surgically removed from each animal. Histopathological examination showed mild-degree infiltration of polymornuclear and mononuclear cells at the interstitium of the KD muscles. Extracted proteins were resolved with two-dimensional electrophoresis (2-DE), and visualized with Coomassie Brilliant Blue R-250 stain. Quantitative intensity analysis revealed 16 up-regulated protein spots in the KD muscles, as compared to the controls. These differentially expressed proteins were subsequently identified by peptide mass fingerprinting and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). Most of the altered proteins induced by chronic potassium depletion were muscle enzymes that play significant roles in several various metabolic pathways. Other up-regulated proteins included myosin-binding protein H, alpha-B Crystallin, and translationally controlled tumor protein (TCTP). These findings may lead to a new roadmap for research on hypokalemic myopathy, to better understanding of the pathophysiology of this medical disease, and to biomarker discovery.

Amino Acid Sequence↗
PubMed2005

Identification of mammalian proteins cross-linked to DNA by ionizing radiation.

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0-4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.

Animals↗
PubMed2004

[Two-dimensional electrophoregram for proteomic analysis of rat brain with intrahippocampal amyloid beta injection and normal rat brain].

OBJECTIVE: To investigate the molecular mechanisms of Alzheimer's disease by comparing global protein patterns in two-dimensional electrophoregram (2-DE) of the brain of rats with intrahippocampal amyloid beta injection and normal rats. METHODS: From adult SD rats with intrahippocampal injection of amyloid beta, 200 microg brain proteins were extracted with 9 mol/L urea, 4% CHAPS, 1% DTT, 0.5% CA and a cocktail of protease inhibitors. Immobilized pH gradient (IPG) isoelectric focusing electrophoresis of the extracted proteins was performed to obtain the electrophoretogram of the first dimension, with the second dimension obtained by vertical SDS-PAGE. The electrophoretograms were visualized using silver staining and analyzed with ImageMaster 2D-Elite software. RESULTS: On average, 496 and 491 protein spots could be obtained in the electrophoregraphs for rats with amyloid beta and the control rats, respectively, and 30 of these spots exhibited quantitative changes. Another 11 and 6 spots were exclusively shown on the protein maps for amyloid beta-treated rats and control rats, respectively. CONCLUSION: The differentially displayed proteins in the brain identified between the rats with intrahippocampal amyloid beta injection and control rats may provide further insight into the pathogenesis of Alzheimer's disease and useful clues for developing new drugs for its treatment.

Alzheimer Disease↗
PubMed2004

Diminished matrix metalloproteinase 9 secretion in human immunodeficiency virus-infected mononuclear phagocytes: modulation of innate immunity and implications for neurological disease.

Neurotoxic secretory products from virus-infected mononuclear phagocytes (MP; perivascular macrophages and microglia) orchestrate the neuropathogenesis of human immunodeficiency virus type one (HIV-1) infection. To uncover such MP products and their relationship to disease, we used a proteomics platform consisting of one dimensional polyacrylamide gel electrophoresis (1-DE), mass spectrometry peptide sequencing, and bioinformatics in order to identify from HIV-1-infected monocyte-derived macrophages (MDM) secretions. Matrix metalloproteinase 9 (MMP 9) secreted in abundance in MDM was markedly down-regulated following viral infection. A negative correlation between MMP 9 and HIV-1 reverse transcriptase activity was shown by quantitative Western blot assays. These data further demonstrate immunoregulatory activities of HIV-1-infected MDM providing unique insights into cellular function in disease.

Blotting, Western↗
PubMed2006

Microbial metalloproteinases mediate sensing of invading pathogens and activate innate immune responses in the lepidopteran model host Galleria mellonella.

Thermolysin-like metalloproteinases such as aureolysin, pseudolysin, and bacillolysin represent virulence factors of diverse bacterial pathogens. Recently, we discovered that injection of thermolysin into larvae of the greater wax moth, Galleria mellonella, mediated strong immune responses. Thermolysin-mediated proteolysis of hemolymph proteins yielded a variety of small-sized (<3 kDa) protein fragments (protfrags) that are potent elicitors of innate immune responses. In this study, we report the activation of a serine proteinase cascade by thermolysin, as described for bacterial lipopolysaccharides (LPS), that results in subsequent prophenoloxidase activation leading to melanization, an elementary immune defense reaction of insects. Quantitative real-time reverse transcription-PCR analyses of the expression of immune-related genes encoding the inducible metalloproteinase inhibitor, gallerimycin, and lysozyme demonstrated increased transcriptional rates after challenge with purified protfrags similar to rates after challenge with LPS. Additionally, we determined the induction of a similar spectrum of immune-responsive proteins that were secreted into the hemolymph by using comparative proteomic analyses of hemolymph proteins from untreated larvae and from larvae that were challenged with either protfrags or LPS. Since G. mellonella was recently established as a valuable pathogenicity model for Cryptococcus neoformans infection, the present results add to our understanding of the mechanisms of immune responses in G. mellonella. The obtained results support the proposed danger model, which suggests that the immune system senses endogenous alarm signals during infection besides recognition of microbial pattern molecules.

Animals↗
PubMed2005

Scale-free networks in cell biology.

A cell's behavior is a consequence of the complex interactions between its numerous constituents, such as DNA, RNA, proteins and small molecules. Cells use signaling pathways and regulatory mechanisms to coordinate multiple processes, allowing them to respond to and adapt to an ever-changing environment. The large number of components, the degree of interconnectivity and the complex control of cellular networks are becoming evident in the integrated genomic and proteomic analyses that are emerging. It is increasingly recognized that the understanding of properties that arise from whole-cell function require integrated, theoretical descriptions of the relationships between different cellular components. Recent theoretical advances allow us to describe cellular network structure with graph concepts and have revealed organizational features shared with numerous non-biological networks. We now have the opportunity to describe quantitatively a network of hundreds or thousands of interacting components. Moreover, the observed topologies of cellular networks give us clues about their evolution and how their organization influences their function and dynamic responses.

Animals↗
PubMed2006

General framework for developing and evaluating database scoring algorithms using the TANDEM search engine.

MOTIVATION: Tandem mass spectrometry (MS/MS) identifies protein sequences using database search engines, at the core of which is a score that measures the similarity between peptide MS/MS spectra and a protein sequence database. The TANDEM application was developed as a freely available database search engine for the proteomics research community. To extend TANDEM as a platform for further research on developing improved database scoring methods, we modified the software to allow users to redefine the scoring function and replace the native TANDEM scoring function while leaving the remaining core application intact. Redefinition is performed at run time so multiple scoring functions are available to be selected and applied from a single search engine binary. We introduce the implementation of the pluggable scoring algorithm and also provide implementations of two TANDEM compatible scoring functions, one previously described scoring function compatible with PeptideProphet and one very simple scoring function that quantitative researchers may use to begin their development. This extension builds on the open-source TANDEM project and will facilitate research into and dissemination of novel algorithms for matching MS/MS spectra to peptide sequences. The pluggable scoring schema is also compatible with related search applications P3 and Hunter, which are part of the X! suite of database matching algorithms. The pluggable scores and the X! suite of applications are all written in C++. AVAILABILITY: Source code for the scoring functions is available from http://proteomics.fhcrc.org

Algorithms↗
PubMed2004

Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome.

Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.

Cells, Cultured↗