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 1,081 records · Page 60Linked to original sources
PubMedDate not supplied

Evaluation of shotgun sequencing for proteomic analysis of human plasma using HPLC coupled with either ion trap or Fourier transform mass spectrometry.

This paper reports on studies directed to the characterization of the proteome of human plasma by the shotgun sequencing approach, namely the use of HPLC coupled to mass spectrometry (MS). The report will present data from two laboratories that allows the comparison of peptide and protein identifications by either accurate mass measurement on a Fourier transform mass spectrometry or MS/MS fragmentation on an ion trap mass spectrometer. Because the dynamic range of the protein components of plasma is one of the largest for a biological sample, the analysis of such a challenging sample was aided by the use of these two MS approaches. The major classes of proteins observed were transport proteins, enzymes, and enzyme inhibitors, blood-clotting factors, membrane-associated proteins including soluble forms of receptors, hormones, immunoglobulins, and other glycoproteins. The protein identifications were also highly consistent with results obtained from 2D gel studies, although a larger number of additional proteins were observed with the shotgun sequencing approach. The quantitation of low to medium level proteins was explored in the ion trap with an add-back of a known amount of human growth hormone (hGH) at a clinically relevant level (5 ug/L). The isotope coded affinity tag (ICAT) approach was used to quantitate successfully different levels of hGH in replicate analysis via the disulfide linked tryptic peptide (T6-T16). These studies suggest that the shotgun sequencing approach can be used to characterize part of the plasma proteome and serve as a starting point for the use of multidimensional analytical approaches for the analysis of complex biological samples.

Algorithms↗
PubMed2006

Proteomics of the heart: unraveling disease.

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in developed countries. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide new insights into cellular mechanisms involved in cardiac dysfunction. The majority of proteomic investigations still use 2D gel electrophoresis (2-DE) with immobilized pH gradients to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. In spite of the development of novel gel-free technologies, 2-DE remains the only technique that can be routinely applied to parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. It can resolve >5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect <1 ng of protein per spot. Furthermore, 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. The use of proteomics to investigate heart disease should result in the generation of new diagnostic and therapeutic markers. In this article, we review the current status of proteomic technologies, describing the 2-DE proteomics workflow, with an overview of protein identification by MS and how these technologies are being applied to studies of human heart disease.

Developed Countries↗
PubMed2005

The proteomics of neurodegeneration.

The continuing improvement and refinement of proteomic and bioinformatic tools has made it possible to obtain increasing amounts of structural and functional information about proteins on a global scale. The emerging field of neuroproteomics promises to provide powerful strategies for further characterizing neuronal dysfunction and cell loss associated with neurodegenerative diseases. Neuroproteomic studies have thus far revealed relatively comprehensive quantitative changes and post-translational modifications (mostly oxidative damage) of high abundance proteins, confirming deficits in energy production, protein degradation, antioxidant protein function, and cytoskeletal regulation associated with neurodegenerative diseases such as Alzheimer and Parkinson disease. The identification of changes in low-abundance proteins and characterization of their functions based on protein-protein interactions still await further development of proteomic methodologies and more dedicated application of these technologies by neuroscientists. Once accomplished, however, the resulting information will certainly provide a truly comprehensive view of neurodegeneration-associated changes in protein expression, facilitating the identification of novel biomarkers for the early detection of neurodegenerative diseases and new targets for therapeutic intervention.

Alzheimer Disease↗
PubMed2002

Single-molecule reader for proteomics and genomics.

Recent developments in ultrasensitive fluorescence microscopy enabled the detection and detailed characterization of individual biomolecules in their native environment. New types of information can be obtained from studying individual molecules, which is not accessible from ensemble measurements. Moreover, this methodological advance matches the need of bioscience to downscale the sample amount required for screening devices. It is envisioned that concentrations as low as approximately 1000 molecules contained in a sample of 1 nl can be detected in a chip-based assay. In this review, we overview state-of-the-art single molecule microscopy with respect to its applicability to ultrasensitive screening. Quantitative estimations will be given, based on a novel apparatus designed for large area screening at single molecule sensitivity.

Genomics↗
PubMed2004

Mass spectrometric approaches for characterizing bacterial proteomes.

The emergence of advanced liquid chromatography mass spectrometry technologies for characterizing very complex mixtures of proteins has greatly propelled the field of proteomics, the goal of which is the simultaneous examination of all the proteins expressed by an organism. This research area represents a paradigm shift in molecular biology by attempting to provide a top-down qualitative and quantitative view of all the proteins (including their modifications and interactions) that are essential for an organism's life cycle, rather than targeting a particular protein family. This level of global protein information about an organism such as a bacterium can be combined with genomic and metabolomic data to enable a systems biology approach for understanding how these organisms live and function.

Bacterial Proteins↗
PubMed2007

Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury.

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among approximately 1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

Analysis of Variance↗
PubMed2006

Development and implementation of a direct detection, quantitation and validation system for class I MHC self-peptide epitopes.

Gene and protein expression studies demonstrate that viral-infected and malignant cells undergo a complex series of transcriptional and translational changes. As class I MHC molecules reflect the proteome (and changes therein) by presenting intracellular peptide epitopes, the development of a direct discovery and validation technology for the identification of these epitopes is needed. We developed our technology using HIV-1-infected cells as a model. A combination of hollow fiber class I HLA protein production and mass spectrometric epitope analysis indicated a 3-fold increase in the host-peptide VLMTEDIKL(720-728), [eIF4G((720))] presented by the HLA-A*0201 of HIV-1-infected cells. This peptide is derived from the host-protein translation of eukaryotic initiation factor 4-gamma (eIF4G) that plays a pivotal role in cellular protein synthesis. Direct confirmation of expression of this self-encoded antigen was performed through development of a T cell receptor mimic (TCRm) monoclonal antibody (mAb). The resulting 4F7 TCRm demonstrated specific recognition of the eIF4G((720))-A*0201 complex. Staining of normal PBMCs with 4F7 showed only low levels of endogenous eIF4G((720)) presentation by HLA-A*0201, while 4F7 staining of HIV-1-infected PBMCs revealed an approximately 3-fold increase in eIF4G((720))-A*0201. The MHC-peptide complex was initially detectable by 4F7 at 3 days post-infection, with a steady increase through day 8. We therefore demonstrate the successful development and implementation of an integrated discovery and validation technology system for direct identification and confirmation of class I MHC-peptide epitopes on cells.

Antibodies, Monoclonal↗
PubMed2004

Proteome analysis reveals elevated serum levels of clusterin in patients with preeclampsia.

Preeclampsia is a pregnancy-specific syndrome and a major cause of maternal mortality. The pathophysiology of preeclampsia is unknown, and no proteome analysis of preeclampsia has been reported. We sought to identify proteins associated with preeclampsia using a proteomic technique and performed two-dimensional electrophoresis (2-DE) on sera from six patients with preeclampsia and six normal pregnant women, followed by comparison of the SYPRO Ruby-stained 2-DE profiles. A group of overexpressed spots was identified in the limited study set. Overexpressed spots were identified as clusterin by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting, a protein database search, and Western blot analysis. Additionally, sera of 80 preeclamptic women and 80 normal pregnant women were processed by immunoassay methods to confirm changes in clusterin concentrations quantitatively. Immunoassays showed that clusterin levels in the 80 preeclamptic women were significantly higher than those in the 80 controls (mean +/- SD; 1.62 +/- 0.46 times reference level in preeclamptic women vs. 1.30 +/- 0.46 times reference level in controls, P < 0.001). Proteomic analysis of serum proteins is a promising tool for studying preeclampsia pathophysiology and identifying proteins associated with preeclampsia.

Blood Proteins↗
PubMed2006

Variation in the holm oak leaf proteome at different plant developmental stages, between provenances and in response to drought stress.

Major proteins of the holm oak leaf proteome have been previously identified using a combination of 2-DE, MS analysis and BLAST similarity search (Jorge et al., Proteomics 2005, 5, 222-234). That study, conducted with field samples from mature trees, revealed the existence of a great variability in the 2-DE protein map, with qualitative as well as quantitative changes, both analytical and biological. A similar study has been carried out with 2-year-old seedlings to analyze and study: (i) changes in the 2-DE protein profile at different tree developmental stages; (ii) the 2-DE protein map variability between three different Spanish provenances; and (iii) variations in the 2-DE protein profile in response to drought stress. Although the protein profile of leaves from seedlings and mature trees was fairly similar, the biological variance found was lower in the former. In the present study, new proteins have been identified. At least four different protein spots differentiated Spanish provenances, two of them identified as an ATP synthase alpha chain, and a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Fourteen different protein spots were qualitatively variable between well-watered and drought-stressed seedlings, with some of them corresponding to enzymes of carbohydrate and protein metabolism. Data presented indicated the mobilization of storage proteins and carbohydrates, as well as photosynthesis inhibition under drought conditions.

Dehydration↗
PubMed2000

Functional genomics by mass spectrometry.

Systematic analysis of the function of genes can take place at the oligonucleotide or protein level. The latter has the advantage of being closest to function, since it is proteins that perform most of the reactions necessary for the cell. For most protein based ('proteomic') approaches to gene function, mass spectrometry is the method of choice. Mass spectrometry can now identify proteins with very high sensitivity and medium to high throughput. New instrumentation for the analysis of the proteome has been developed including a MALDI hybrid quadrupole time of flight instrument which combines advantages of the mass finger printing and peptide sequencing methods for protein identification. New approaches include the isotopic labeling of proteins to obtain accurate quantitative data by mass spectrometry, methods to analyze peptides derived from crude protein mixtures and approaches to analyze large numbers of intact proteins by mass spectrometry directly. Examples from this laboratory illustrate biological problem solving by modern mass spectrometric techniques. These include the analysis of the structure and function of the nucleolus and the analysis of signaling complexes.

Genome↗
PubMed2005

Proteomic analysis of Rickettsia prowazekii.

Rickettsia prowazekii is an obligate intracellular gram-negative bacterium. Comparative proteomics study of a virulent strain (Breinl) versus an avirulent strain (Madrid E) was performed using an integrated liquid chromatography and mass spectrometer. About 30% of predicted proteins were detected and identified. Among the detected proteins, more than 30 proteins were of unknown function in both strains. Although several proteins were detected in only one strain, the overall distribution of detected proteins in different COGs (clusters of orthologs groups) was very similar between the two strains. Functional analysis of differentially expressed proteins, either qualitatively or quantitatively, may lead to the discovery of pathogenesis-related factors.

Bacterial Proteins↗
PubMed2006

Proteomic and computational methods in systems modeling of cellular signaling.

Cellular signaling lies at the core of cellular behavior, and is central for the understanding of many pathologic conditions. To comprehend how signal transduction is orchestrated at the molecular level remains the ultimate challenge for cell biology. In the last years there has been a revolution in the development of high-throughput methodologies in proteomics and genomics, which have provided extensive knowledge about expression profiles and molecular interaction-networks. However, these methods have typically provided qualitative and static information. This is about to turn, and several high-throughput methods are now available that provide quantitative and temporal information. These data are well suited for analysis by computational methods and bioinformatics, which are becoming increasingly valuable tools to grasp the complexity of cellular networks. At present, several cellular pathways have been modeled in silico and the analysis provides new understanding of the underlying properties that contribute to their dynamic features. Here, we review methodologies that are used for in silico modeling as well as methods to obtain large-scale quantitative data, and discuss how they can be integrated to generate powerful and predictive models of cellular processes. We argue that the generation of such models provide powerful tools to understand how systems properties emerges in healthy and pathologic states, and to generate efficient strategies for pharmacological intervention.

Computational Biology↗
PubMed2005

Comparative proteomic analysis provides new insights into chilling stress responses in rice.

Low temperature is one of the major abiotic stresses limiting the productivity and the geographical distribution of many important crops. To gain a better understanding of chilling stress responses in rice (Oryza sativa L. cv. Nipponbare), we carried out a comparative proteomic analysis. Three-week-old rice seedlings were treated at 6 degrees C for 6 or 24 h and then recovered for 24 h. Chilling treatment resulted in stress phenotypes of rolling leaves, increased relative electrolyte leakage, and decreased net photosynthetic rate. The temporal changes of total proteins in rice leaves were examined using two-dimensional electrophoresis. Among approximately 1,000 protein spots reproducibly detected on each gel, 31 protein spots were down-regulated, and 65 were up-regulated at least at one time point. Mass spectrometry analysis allowed the identification of 85 differentially expressed proteins, including well known and novel cold-responsive proteins. Several proteins showed enhanced degradation during chilling stress, especially the photosynthetic proteins such as Rubisco large subunit of which 19 fragments were detected. The identified proteins are involved in several processes, i.e. signal transduction, RNA processing, translation, protein processing, redox homeostasis, photosynthesis, photorespiration, and metabolisms of carbon, nitrogen, sulfur, and energy. Gene expression analysis of 44 different proteins by quantitative real time PCR showed that the mRNA level was not correlated well with the protein level. In conclusion, our study provides new insights into chilling stress responses in rice and demonstrates the advantages of proteomic analysis.

Amino Acid Sequence↗
PubMed2006

OncoDB.HCC: an integrated oncogenomic database of hepatocellular carcinoma revealed aberrant cancer target genes and loci.

The OncoDB.HCC (http://oncodb.hcc.ibms.sinica.edu.tw) is based on physical maps of rodent and human genomes containing quantitative trait loci of rodent HCC models and various human HCC somatic aberrations including chromosomal data from loss of heterozygosity and comparative genome hybridization analyses, altered expression of genes from microarray and proteomic studies, and finally experimental data of published HCC genes. Comprehensive integration of HCC genomic aberration data avoids potential pitfalls of data inconsistency from single genomic approach and provides lines of evidence to reveal somatic aberrations from levels of DNA, RNA to protein. Twenty-nine of 30 (96.7%) novel HCC genes with significant altered expressions in compared between tumor and adjacent normal tissues were validated by RT-PCR in 45 pairs of HCC tissues and by matching expression profiles in 57 HCC patients of re-analyzed Stanford HCC microarray data. Comparative mapping of HCC loci in between human aberrant chromosomal regions and QTLs of rodent HCC models revealed 12 syntenic HCC regions with 2 loci effectively narrowed down to 2 Mb. Together, OncoDB.HCC graphically presents comprehensive HCC data integration, reveals important HCC genes and loci for positional cloning and functional studies, and discloses potential molecular targets for improving HCC diagnosis and therapy.

Animals↗
PubMed2004

GenomeViz: visualizing microbial genomes.

BACKGROUND: An increasing number of microbial genomes are being sequenced and deposited in public databases. In addition, several closely related strains are also being sequenced in order to understand the genetic basis of diversity and mechanisms that lead to the acquisition of new genetic traits. These exercises have necessitated the requirement for visualizing microbial genomes and performing genome comparisons on a finer scale. We have developed GenomeViz to enable rapid visualization and subsequent comparisons of several microbial genomes in an interactive environment. RESULTS: Here we describe a program that allows visualization of both qualitative and quantitative information from complete and partially sequenced microbial genomes. Using GenomeViz, data deriving from studies on genomic islands, gene/protein classifications, GC content, GC skew, whole genome alignments, microarrays and proteomics may be plotted. Several genomes can be visualized interactively at the same time from a comparative genomic perspective and publication quality circular genome plots can be created. CONCLUSIONS: GenomeViz should allow researchers to perform visualization and comparative analysis of up to eight different microbial genomes simultaneously.

Bacterial Proteins↗
PubMed2006

Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein.

BACKGROUND: Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. METHODS: Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. RESULTS: 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. CONCLUSION: ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

Biomarkers, Tumor↗
PubMed2004

Mapping phosphorylation sites: a new strategy based on the use of isotopically labelled DTT and mass spectrometry.

Phosphoproteomics, nowadays, represents a front line in functional proteomics as testified by the number of papers recently appearing in the literature. In an attempt to improve and simplify the methods so far suggested we have set up a simple isotope-coded approach to label and quantitate phospho-Ser/-Thr residues in protein mixtures. First of all, after appropriate oxidation of cysteine/cystine residues followed by tryptic hydrolysis, we have optimised and simplified the beta-elimination reaction to get the corresponding alkene moiety from the phosphate esters. This was achieved by (a) separating the elimination reaction from the addition reaction, (b) the use of Ba(OH)(2) as alkali reagent and (c) its further elimination by the simple addition of solid CO(2) to the peptide mixture. The Michael reaction was then performed, after the removal of BaCO(3) by centrifugation, by adding dithiothreitol (DTT) to the peptide mixture. Finally, the direct purification of the modified phosphopeptides was performed on a thiol-sepharose column. The availability of fully deuterated DTT, introducing a 6 Da difference with respect to the non-deuterated species, allows quantitation of the differential extent of signalling modification when analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and liquid chromatography/mass spectrometry. The entire procedure has been set up by using bovine alpha-casein, and resulted in the identification of all the phosphorylated tryptic peptides, including the tetraphosphorylated peptides, which escaped all previously reported procedures

Amino Acid Sequence↗
PubMed2006

Molecular genetic studies of gene identification for osteoporosis: a 2004 update.

This review summarizes comprehensively the most important and representative molecular genetics studies of gene identification for osteoporosis published up to the end of December 2004. It is intended to constitute a sequential update of our previously published review covering the available data up to the end of 2002. Evidence from candidate gene association studies and genome-wide linkage studies in humans, as well as quantitative trait locus mapping animal models are reviewed separately. Studies of transgenic and knockout mice models relevant to osteoporosis are summarized. An important extension of this update is incorporation of functional genomic studies (including DNA microarrays and proteomics) on osteogenesis and osteoporosis, in light of the rapid advances and the promising prospects of the field. Comments are made on the most notable findings and representative studies for their potential influence and implications on our present understanding of genetics of osteoporosis. The format adopted by this review should be ideal for accommodating future new advances and studies.

Animals↗