Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “targeted 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 37 records · Page 2Linked to original sources
PubMed2005

Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer.

To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.

14-3-3 Proteins↗
PubMed2002

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

Amino Acid Sequence↗
PubMed2006

Targeted comparative proteomics by liquid chromatography/matrix-assisted laser desorption/ionization triple-quadrupole mass spectrometry.

Here we report the first application of a matrix-assisted laser desorption/ionization (MALDI) triple-quadrupole mass spectrometer for targeted proteomics. Employing an amine-specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n = 5) and a detection limit of 14 amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two-dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D-LC/ESI)-MS/MS. Using LC/MALDI-MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7 +/- 0.8.

Amino Acid Sequence↗
PubMed2024

High-Throughput and High-Sensitivity Biomarker Monitoring in Body Fluid by Fast LC SureQuant IS-Targeted Quantitation.

Targeted proteomics methods have been greatly improved and refined over the last decade and are becoming increasingly the method of choice in protein and peptide quantitative assays. Despite the tremendous progress, targeted proteomics assays still suffer from inadequate sensitivity for lower abundant proteins and throughput, especially in complex biological samples. These attributes are essential for establishing targeted proteomics methods at the forefront of clinical use. Here, we report an assay utilizing the SureQuant internal standard-triggered targeted method on a latest generation mass spectrometer coupled with an EvoSep One liquid chromatography platform, which displays high sensitivity and a high throughput of 100 samples per day. We demonstrate the robustness of this method by quantifying proteins spanning six orders of magnitude in human wound fluid exudates, a biological fluid that exhibits sample complexity and composition similar to plasma. Among the targets quantified were low-abundance proteins such at tumor necrosis factor A and interleukin 1-β, highlighting the value of this method in the quantification of trace amounts of invaluable biomarkers that were until recently hardly accessible by targeted proteomics methods. Taken together, this method extends the toolkit of targeted proteomics assays and will help to drive forward mass spectrometry-based proteomics biomarker quantification.

Humans↗
PubMed2004

A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.

Adenine↗
PubMed2004

Differential polypeptide display: the search for the elusive target.

Proteomics, as a tool to identify proteins in biological samples, is gaining rapidly importance in the postgenomic era. Here we discuss the current and potential role of different techniques in the field of proteomics such as two-dimensional gel electrophoresis off-line coupled to MALDI-MS (2D-PAGE-MALDI-MS), high performance liquid chromatography mass spectrometry (HPLC-MS), surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) and a newly developed technique, capillary electrophoresis mass spectrometry (CE-MS). The developments of the last years are presented discussed.

Chromatography, High Pressure Liquid↗
PubMed2026

Both L-Lactyl and D-Lactyl Enantiomers Modify Histones in Mouse Testis.

Dynamic histone posttranslational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the former may more likely modify histones due to abundant L-lactate produced by glycolysis. Here, we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, despite the fact that L-lactate is much more abundant than D-lactate in mouse testis, we estimated abundance ratios of L-over D-lactylation to lie between 0.4 and 1.6 on seven residues of histones H3 and H4. Next, targeted proteomic analyses were performed on histones extracted from meiotic and postmeiotic male germ cells (spermatocytes and round spermatids, respectively), which are known to use L-lactate as a main source of energy. Nonetheless, residues 18 and 23 of histone H3 (H3K18 and H3K23) were reliably quantified and shown to harbor balanced amounts of both enantiomers. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing about 0.01 to 0.44%: this contrasts with acetylation which exists at up to 25 to 35% relative abundances on some N-terminal lysines. Yet, lactylation appears to be more abundant than acetylation on the C-terminal half of H3 and H4, where the latter modification is scarce. Collectively, our results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, that leads to the modification of histones by L- and D-lactylation.

Animals↗
PubMed2004

Chemical dimerizers and three-hybrid systems: scanning the proteome for targets of organic small molecules.

The integration of technological advances in areas as diverse as chemical biology, proteomics, genomics, automation, and bioinformatics has led to the emergence of novel screening paradigms for analyzing the molecular basis of drug action. This review summarizes recent advances in three-hybrid technologies and their application to the characterization of small molecule-protein interactions and proteome-wide identification of drug receptors.

Animals↗
PubMed2006

Putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes.

Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes.

Animals↗
PubMed2005

Silicone/graphite coating for on-target desalting and improved peptide mapping performance of matrix-assisted laser desorption/ionization-mass spectrometry targets in proteomic experiments.

In two-dimensional gel electrophoresis-based proteomic experiments matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting is often the technique of choice in identifying proteins. Here, we present a novel surface coating technique for MALDI-MS targets that improves manual and automatic sample analysis. A mixture of silicone and graphite is spread in the form of a thin layer over the target. Due to the hydrophobicity of the coating, aqueous solutions can be applied to relatively small spots very precisely using a robotic system. At least four times more liquid can be concentrated on the same area compared to uncoated steel targets. alpha-cyano-4-hydrocinnamic acid crystallizes in form of very small crystals evenly distributed over the surface. The search for "hot spots" during the analysis is not necessary, which supports the automatic acquisition of data. The homogeneous crystal layer can be very effectively washed on-target without encountering major sample losses. This efficient washing and the focused application of aqueous samples replace expensive and time-consuming reversed phase micro column based sample clean-ups. When analyzing peptide mixtures, the signal intensities are up to five times higher than with preparations of the same un-desalted samples on steel targets, since four times more sample can be loaded. The mass resolution remains unaffected by the surface coating. After usage the coating can be removed, followed by a new coating avoiding any carry-over of sample to the next analysis. All these properties make the precoating of MALDI-MS targets with a silicone/graphite layer an ideal technique for routine analysis in large-scale proteomic experiments.

Autoanalysis↗
PubMed2005

Targeted comparative proteomics by liquid chromatography-tandem Fourier ion cyclotron resonance mass spectrometry.

In proteomics, effective methods are needed for identifying the relatively limited subset of proteins displaying significant changes in abundance between two samples. One way to accomplish this task is to target for identification by MS/MS only the "interesting" proteins based on the abundance ratio of isotopically labeled pairs of peptides. We have developed the software and hardware tools for online LC-FTICR MS/MS studies in which a set of initially unidentified peptides from a proteome analysis can be selected for identification based on their distinctive changes in abundance following a "perturbation". We report here the validation of this method using a mixture of standard proteins combined in different ratios after isotopic labeling. We also demonstrate the application of this method to the identification of Shewanella oneidensis peptides/proteins exhibiting differential abundance in suboxic versus aerobic cell cultures.

Bacterial Proteins↗
PubMed2003

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors.

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.

14-3-3 Proteins↗
PubMed2003

Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes.

In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types.

Animals↗
PubMed2002

Proteomics: drug target discovery on an industrial scale.

The discovery of targets that are sufficiently robust to yield marketable therapeutics is an enormous challenge. Through the years, several approaches have been used with varying degrees of success. These include target-independent screening of tumor-derived cell lines (disease-dependent), reductionist approaches to identifying crucial elements of disease-affected pathways, disease-independent screening of homologs of previously drugged targets, disease-dependent 'global' examination of gene transcript levels, and disease-dependent global examination of protein expression levels. These endeavors have been enabled by several major advancements in technology, most recently, the sequencing of the human genome. This review identifies the technical issues to be addressed for industrial-scale protein-based discovery in the identification of targets for therapeutic (or diagnostic) intervention. Such approaches aim to direct discovery in a way that increases the probability of robust target identification, and decreases the probability of failure owing to variable expression in this emerging field.

Algorithms↗
PubMed2004

Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.

Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

Adipocytes↗
PubMed2006

Global and targeted quantitative proteomics for biomarker discovery.

The extraordinary developments made in proteomic technologies in the past decade have enabled investigators to consider designing studies to search for diagnostic and therapeutic biomarkers by scanning complex proteome samples using unbiased methods. The major technology driving these studies is mass spectrometry (MS). The basic premises of most biomarker discovery studies is to use the high data-gathering capabilities of MS to compare biological samples obtained from healthy and disease-afflicted patients and identify proteins that are differentially abundant between the two specimen. To meet the need to compare the abundance of proteins in different samples, a number of quantitative approaches have been developed. In this article, many of these will be described with an emphasis on their advantageous and disadvantageous for the discovery of clinically useful biomarkers.

Biomarkers↗
PubMed2024

Comprehensive Proteomic Analysis Reveals Distinct Features and a Diagnostic Biomarker Panel for Early Pregnancy Loss in Histological Subtypes.

Early pregnancy loss (EPL) is a common event in human reproduction and is classified into histological subtypes such as hydropic abortion (HA) and hydatidiform moles, including complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs). However, accurate diagnosis and improved patient management remain challenging due to high rates of misdiagnosis and diverse prognostic risks. Therefore, diagnostic biomarkers for EPL are urgently needed. Our study aimed to identify biomarkers for EPL through comprehensive proteomic analysis. Ten CHMs, six PHMs, ten HAs, and 10 normal control products of conception were used to obtain a proteomic portrait. Parallel reaction monitoring-targeted proteomic and regression analyses were used to verify and select the diagnostic signatures. Finally, 14 proteins were selected and a panel of diagnostic classifiers (DLK1, SPTB/COL21A1, and SAR1A) was built to represent the CHM, PHM, and normal control groups (area under the receiver operating characteristic curve = 0.900, 0.804/0.885, and 0.991, respectively). This high diagnostic power was further validated in another independent cohort (n = 148) by immunohistochemistry (n = 120) and Western blot analyses (n = 28). The protein SPTB was selected for further biological behavior experiments in vitro. Our data suggest that SPTB maintains trophoblast cell proliferation, angiogenesis, cell motility, and the cytoskeleton network. This study provides a comprehensive proteomic portrait and identifies potential diagnostic biomarkers. These findings enhance our understanding of EPL pathogenesis and offer novel targets for diagnosis and therapeutic interventions.

Humans↗