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At least 577 records · Page 32Linked to original sources
PubMed2026

Inhibitory mechanism of anthocyanin B-ring substituents on advanced glycation end-product formation through bovine serum albumin binding: Insights from multispectral, molecular docking and proteomics approaches.

This study demonstrated that the inhibitory effect of anthocyanins on AGEs formation is highly dependent on the substitution pattern of the B-ring. Among the four anthocyanins, delphinidin-3-O-glucoside (D3G) exhibited the most potent antiglycation activity across BSA-fructose, MGO, and GO models with half-maximal inhibitory concentration (IC50) of 30.77, 200.29 and 269.97 μM. This superior performance was attributed to the presence of three hydroxyl groups on the B-ring, which facilitates a high-affinity, spontaneous binding interaction with BSA primarily through hydrophobic forces and hydrogen bonding. Spectroscopic and computational analyses revealed that D3G effectively stabilizes the protein scaffold, specifically recovering α-helix content and shielding critical subdomains (IB, IIA, and IIIA). Proteomics data are consistent with a protective binding mechanism, suggesting that D3G reduces the accessibility of key lysine and arginine residues to glycation-induced modifications. These findings provide a structural basis for developing D3G-rich extracts as targeted, structure-based functional ingredients to mitigate glycation-associated food quality degradation and related health issues.

Anthocyanins↗
PubMed2004

Utilization of a new biotinylation reagent in the development of a nondiscriminatory investigative approach for the study of cell surface proteins.

In order to circumvent the various problems encountered during the study of membrane-bound proteins, we designed and synthesized a novel membrane-impermeable biotinylation reagent incorporating chemical properties compatible with this goal. We then developed a nondiscriminatory analytical procedure for such studies which overcomes possible selectivity, contamination and solubility problems. The necessary steps (labeling, limited in situ proteolysis, affinity purification) are all conducted in mild or near native conditions. This versatile method could provide an accurate picture of the cell surface proteome.

Animals↗
PubMed2004

Zooming in: fractionation strategies in proteomics.

The recent development of mass spectrometry, i.e., high sensitivity, automation of protein identification and some post-translational modifications (PTMs) significantly increased the number of large-scale proteomics projects. However, there are still considerable limitations as none of the currently available proteomics techniques allows the analysis of an entire proteome in a single step procedure. On the other hand, there are several successful studies analyzing well defined groups of proteins, e.g., proteins of purified organelles, membrane microdomains or isolated proteins with certain PTMs. Coupling of advanced separation methodologies (different prefractionation strategies, such as subcellular fractionation, affinity purification, fractionation of proteins and peptides according to their physicochemical properties) to highly sensitive mass spectrometers provides powerful means to detect and analyze dynamic changes of low abundant regulatory proteins in eukaryotic cells on the subcellular level. This review summarizes and discusses recent strategies in proteomics approaches where different fractionation strategies were successfully applied.

Animals↗
PubMed2005

Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies.

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.

Acidithiobacillus↗
PubMed2003

Affinity capture of specific DNA-binding proteins for mass spectrometric identification.

We describe a general approach for affinity microcapture of site-specific, nucleic acid-binding proteins. The major difficulties to developing this method into a widely applicable protocol derived from the need for a massive enrichment and the inadvertent, extensive binding of nonspecific proteins to the bait. On the basis of a detailed analysis, we propose (i) a one-step fractionation of crude extracts on P11 phosphocellulose, followed by (ii) a discrete series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format, with cobalt magnets, concatamerized and biotinylated ligands, selective salt conditions, and improved competitor DNAs. We also present rules for determining the precise number and order of selections. The approach and protocol allowed isolation of four, low-abundance transcription factors and repressors from 2 x 10(9) cultured leukemia cells. Captured proteins were 10-20,000-fold enriched from the nuclear extract, in a form and amounts that permitted facile MALDI-TOF and TOF/TOF MS-based protein identification. This is 1-2 orders of magnitude better than many previous efforts and in a fraction of the time (approximately 1 factor/week). The method can be applied to any protein that binds DNA, including those with modest to low affinity, and bridges functional-biochemical studies on replication, transcriptional regulation, and DNA repair with the analytical power of mass spectrometry-based proteomics.

Cell Line, Tumor↗
PubMed2004

Recent advances in chemical genomics.

Chemical genomics, which utilizes specially designed small chemical compounds early in the discovery phase of new drugs to explore the life science at various levels, can address biological questions that are not amenable to genetic manipulation or functional genomics/proteomics approaches. Following the development of HT phenotypic assays and DNA expression analysis, the integration of cell-based assays with activity / affinity-based approaches allows us to interrogate the cells by analyzing phenotypic alterations, changes of transcript signature or detecting the differences in protein expression levels. Furthermore, activity / affinity-based techniques directly provide a druggable subset of gene products, which interact with small molecules, greatly reducing the complexity of analyzing the proteome. In this paper, we give an account of the recent advances (approaches and strategies) in the field of chemical genomics, and discuss how these approaches enable the investigator to obtain a novel therapeutically relevant target as well as drug candidates acting on them in a target-specific manner. This novel post-genomic discovery strategy, where target identification/ validation is carried out by interactions with small molecules, could significantly reduce the time-scale for early drug discovery, and increase the success rate of finding novel, druggable targets, as well as more specific drug candidates.

Animals↗
PubMed2005

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis.

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.

Amino Acid Sequence↗
PubMed2007

Lysosomal membranes from beige mice contain higher than normal levels of endoplasmic reticulum proteins.

Chediak-Higashi syndrome is characterized by dysfunctional giant organelles of common origin, that is, lysosomes, melanosomes, and platelet dense bodies. Its defective gene LYST encodes a large molecular weight protein whose function is unknown. The Beige mouse also defective in Lyst is a good model of the human disease. Purified lysosomes from Beige and normal black mouse livers were used to carry out a proteomics study. Two-dimensional gel electrophoretic separation of soluble lysosomal proteins of Beige and normal mice revealed no major differences. The cleavable isotope-coded affinity tag (cICAT) technique was used to compare the composition of Beige and normal lysosomal membrane proteins. While the levels of common proteins, that is, Lamp1, Lamp2, and Niemann-Pick type C1, were decreased in Beige mice, there was an increase in the levels of endoplasmic reticulum (ER) resident proteins, for example, cytochrome P450, NADPH-cytochrome P450 oxidoreductase, and flavin-containing monooxygenase. Confocal microscopy confirmed that another ER protein, calnexin, colocalizes with Lamp1 on membranes of giant lysosomes from fibroblasts of Chediak-Higashi syndrome patient. Our results suggest that LYST may play a role in either preventing inappropriate incorporation of proteins into the lysosomal membrane or in membrane recycling/maturation.

Animals↗
PubMed2006

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Activated Protein C Resistance↗
PubMed2005

Mass spectrometry for protein identification and the study of post translational modifications.

Mass spectrometry is a powerful tool for the structural characterization of proteins. The development of a large number of analytical strategies based on different mass spectrometric techniques has established mass spectrometry as an indispensable technology to interpret the information encoded in the genome. This manuscript intends to provide a rapid view of principles and instrumentation in mass spectrometry-based proteomics, together with a description of the basic features and limits of the analysis of post translational modifications by mass spectrometry. The application of one of the available mass spectrometry technologies for protein identification to a multi-protein complex isolated in a specific functional context is reported. The identification of a phosphorylation site on a single, purified protein is also illustrated as an example.

Animals↗
PubMed2006

Analysis of the cGMP/cAMP interactome using a chemical proteomics approach in mammalian heart tissue validates sphingosine kinase type 1-interacting protein as a genuine and highly abundant AKAP.

The cyclic nucleotide monophosphates cAMP and cGMP play an essential role in many signaling pathways. To analyze which proteins do interact with these second messenger molecules, we developed a chemical proteomics approach using cAMP and cGMP immobilized onto agarose beads, via flexible linkers in the 2- and 8-position of the nucleotide. Optimization of the affinity pull-down procedures in lysates of HEK293 cells revealed that a large variety of proteins could be pulled down specifically. Identification of these proteins by mass spectrometry showed that many of these proteins were indeed genuine cAMP or cGMP binding proteins. However, additionally many of the pulled-down proteins were more abundant AMP/ADP/ATP, GMP/GDP/GTP, or general DNA/RNA binding proteins. Therefore, a sequential elution protocol was developed, eluting proteins from the beads using solutions containing ADP, GDP, cGMP, and/or cAMP, respectively. Using this protocol, we were able to sequentially and selectively elute ADP, GDP, and DNA binding proteins. The fraction left on the beads was further enriched, for cAMP/cGMP binding proteins. Transferring this protocol to the analysis of the cGMP/cAMP "interactome" in rat heart ventricular tissue enabled the specific pull-down of known cAMP/cGMP binding proteins such as cAMP and cGMP dependent protein kinases PKA and PKG, several phosphodiesterases and 6 AKAPs, that interact with PKA. Among the latter class of proteins was the highly abundant sphingosine kinase type1-interating protein (SKIP), recently proposed to be a potential AKAP. Further bioinformatics analysis endorses that SKIP is indeed a genuine PKA interacting protein, which is highly abundant in heart ventricular tissue.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Proteomic analysis of complexes formed by human topoisomerase I.

Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.

DNA Topoisomerases, Type I↗
PubMed2005

Quantitative analysis of severe acute respiratory syndrome (SARS)-associated coronavirus-infected cells using proteomic approaches: implications for cellular responses to virus infection.

We present the first proteomic analysis on the cellular response to severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. The differential proteomes of Vero E6 cells with and without infection of the SARS-CoV were resolved and quantitated with two-dimensional differential gel electrophoresis followed by ESI-MS/MS identification. Moreover isotope-coded affinity tag technology coupled with two-dimensional LC-MS/MS were also applied to the differential proteins of infected cells. By combining these two complementary strategies, 355 unique proteins were identified and quantitated with 186 of them differentially expressed (at least 1.5-fold quantitative alteration) between infected and uninfected Vero E6 cells. The implication for cellular responses to virus infection was analyzed in depth according to the proteomic results. Thus, the present work provides large scale protein-related information to investigate the mechanism of SARS-CoV infection and pathogenesis.

Amino Acid Sequence↗
PubMed2002

Proteomics for cancer biomarker discovery.

The emergence of novel technologies allows researchers to facilitate the comprehensive analyses of genomes, transcriptomes, and proteomes in health and disease. The information that is expected from such technologies may soon exert a dramatic change in the pace of cancer research and impact dramatically on the care of cancer patients. These approaches have already demonstrated the power of molecular medicine in discriminating among disease subtypes that are not recognizable by traditional pathologic criteria and in identifying specific genetic events involved in cancer progression. This review covers a selection of advances in the realm of proteomics and its promise for cancer biomarker discovery. It also addresses issues regarding sample preparation and specificity and discusses current challenges that need to be overcome. Finally, the review touches on the efforts of the Early Detection Research Network at the National Cancer Institute in promoting biomarker discovery for translation at the clinical level.

Affinity Labels↗
PubMed2004

Optimization of reversed-phase microcapillary liquid chromatography for quantitative proteomics.

Currently, the field of shotgun proteomics relies primarily on the separation of peptides by reversed-phase microcapillary chromatography (RP-microLC) combined with either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS) for protein identification as well as quantification. For this purpose we herein describe construction of a RP-microLC-ESI column-emitter along with optimized microLC conditions for using the device to quantify pair-wise changes in protein expression via the isotope coded affinity tag (ICAT) method that also maximize peak capacity. These optimized RP-microLC parameters required a balance be reached between the disparate needs of quantification which requires good peak shape and identification (i.e. proteome coverage) of proteins via peptide collision induced dissociation (CID) which requires peak capacity be maximized. A complex biological sample from a study of murine acetaminophen toxicity in hepatocyes was chosen for method development because of the high level complexity, but the biological results are not the focus of this manuscript.

Amino Acid Sequence↗
PubMed2001

Phosphoamino acid analysis.

Phosphorylation of amino acid residues in proteins plays a major role in biological systems. Often, phosphorylation acts as a molecular switch controlling the protein activity in different pathways as in metabolism, signal transduction, cell division etc. Therefore, identification of phosphoamino acids in proteins is an important task in protein analysis. Since the introduction of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) a separation of phosphorylated and dephosphorylated protein species is possible. The identification of phosphorylation sites from preparative 2-D gels need very sensitive mass spectrometry methods and a specific enrichment of the phosphoprotein or -peptide.

Amino Acid Sequence↗
PubMed2004

Quantitative protein profiling using antibody arrays.

Traditional approaches to microarrays rely on direct binding assays where the extent of hybridisation and the signal detected are a measure of the analyte concentration in the experimental sample. This approach, directly imported from the nucleic acid field, may fail if applied to antibody-antigen interactions due to the shortage of characterised antibodies, the significant heterogeneity of antibody affinities, their dependence on the extent of protein modification during labelling and the inherent antibody cross-reactivity. These problems can potentially limit the multiplexing capabilities of protein affinity assays and in many cases rule out quantitative protein profiling using antibody microarrays. A number of approaches aimed at achieving quantitative protein profiling in a multiplex format have been reported recently. Of those reported, the three most promising routes include signal amplification, multicolour detection and competitive displacement approaches to multiplex affinity assays. One in particular, competitive displacement, also overcomes the problems associated with quantitation of affinity interactions and provides the most generic approach to highly parallel affinity assays, including antibody arrays.

Animals↗
PubMed2005

A study of glycoproteins in human serum and plasma reference standards (HUPO) using multilectin affinity chromatography coupled with RPLC-MS/MS.

The glycoproteome is a major subproteome present in human plasma. In this study, we isolated and characterized approximately 150 glycoproteins from the human plasma and serum samples provided by HUPO using a multilectin affinity column. The corresponding tryptic digest was separated by RP-HPLC coupled to an IT mass spectrometer (3-D LCQ). Also in this study, a new system, namely an Ettan MDLC system coupled to a linear ITLTQ, was compared with the previous LCQ platform and gave a greater number of protein identifications, as well as better quality. When we compared the composition of the glycoproteomes for the plasma and serum samples there was a close correlation between the samples, except for the absence of fibrinogen from the identified-protein list in the latter sample, which was presumably as a result of the clotting process. In addition, the analysis of the samples from three ethnic specimens, Caucasian American, Asian American, and African American, were very similar but showed a higher angiotensinogen plasma level and a lower histidine-rich glycoprotein level in Caucasian American samples, and a lower vitronectin level in African American blood samples.

Blood Proteins↗