Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “affinity 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 199 records · Page 11Linked to original sources
PubMed2025

High-affinity CD16A polymorphism associated with reduced risk ofsevere COVID-19.

CD16A is an activating Fc receptor on NK cells that mediates antibody-dependent cellular cytotoxicity (ADCC), a key mechanism in antiviral immunity. However, the role of NK cell-mediated ADCC in SARS-CoV-2 infection remains unclear, particularly whether it limits viral spread and disease severity or contributes to the immunopathogenesis of COVID-19. We hypothesized that the high-affinity CD16AV176 polymorphism influences these outcomes. Using an in vitro reporter system, we demonstrated that CD16AV176 is a more potent and sensitive activator than the common CD16AF176 allele. To assess its clinical relevance, we analyzed 1,027 patients hospitalized with COVID-19 from the Immunophenotyping Assessment in a COVID-19 cohort (IMPACC), a comprehensive longitudinal dataset with extensive transcriptomic, proteomic, and clinical data. The high-affinity CD16AV176 allele was associated with a significantly reduced risk of ICU admission, mechanical ventilation, and severe disease trajectories. Lower anti-SARS-CoV-2 IgG titers were correlated to CD16AV176; however, there was no difference in viral load across CD16A genotypes. Proteomic analysis revealed that participants homozygous for CD16AV176 had lower levels of inflammatory mediators. These findings suggest that CD16AV176 enhances early NK cell-mediated immune responses, limiting severe respiratory complications in COVID-19. This study identifies a protective genetic factor against severe COVID-19, informing future host-directed therapeutic strategies.

Humans↗
PubMed2004

Profiling the activity of G proteins in patient-derived tissues by rapid affinity-capture of signal transduction proteins (GRASP).

The next phase in molecular medicine will require the ability to identify signal transduction events inside a cell, in the biologic context of the disease-host interface and at a given point in time. New technologies are needed to profile the activity of these signaling pathways in patient tissue rather than cultured cell lines since the tumor-host microenvironment influences the cellular proteome. We introduce such a technology, rapid affinity capture of signaling proteins (GRASP), to investigate the activity of signaling pathways from patient-derived carcinomas and benign epithelial surfaces and apply it to studying important signaling events in ovarian carcinoma. During the progression from benign ovarian epithelium to invasive carcinoma, there is loss of repression of Rho A as evidenced by its dissociation from its inhibitor, Rho Guanine Nucleotide Dissociation Inhibitor (RhoGDI). GRASP is more informative than simply profiling transcript or protein levels. Furthermore, GRASP coupled with mass spectrometry allowed us to identify a protein-binding partner of RhoGDI, demonstrating the power of this technology in the discovery of potentially novel protein-protein interactions. GRASP represents an advance in the field of proteomics as it detects protein interactions present in cells as they exist in their native tissue microenvironment.

Carcinoma↗
PubMed2002

Two-dimensional gel electrophoresis; better than a poke in the ICAT?

To date, the most widely used technology for conducting proteomic studies has been two-dimensional gel electrophoresis (2DGE), but this approach does have drawbacks. Isotope-coded affinity tagging (ICAT) is starting to challenge 2DGE as a new proteomic tool for the analysis of proteins in complex biological specimens. An appraisal of these two methodologies reveals that neither ICAT nor 2DGE provide comprehensive coverage on a proteome-wide scale.

Chromatography, Affinity↗
PubMed2003

Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography.

Bone is maintained by two cell types, bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts express two factors, osteoprotegerin and receptor activator of NF-kappaB ligand (RANKL), inhibiting and promoting osteoclast differentiation, respectively. In contrast, modulators of bone resorption expressed by osteoclasts have not been so well studied enough. In the present study, we demonstrate proteome analysis of secreted proteins during osteoclast differentiation to elucidate the molecular mechanism of bone resorption and bone remodeling. To achieve this objective, we chose RAW264.7 cells with RANKL as a homogeneous osteoclast differentiation model and used two methods, two-dimensional gel electrophoresis (2-DE) and isotope-coded affinity tags (ICAT) analysis with two-dimensional liquid chromatography. We found 23 spots in 2-DE and 19 proteins in ICAT analysis which were expressed differently during osteoclast differentiation. These two methods gave us closely related but different information about proteins, suggesting they are complementary or at least supplementary methods at present. Cathepsins, osteopontin, legumain, macrophage inflammatory protein-1alpha, and other proteins were observed as up- or down-regulated proteins and are discussed in the context of osteoclast differentiation and bone resorption. In addition to confirming previous observations, this study indicates novel proteins related to osteoclast differentiation which are potential therapeutic targets for the treatment of bone diseases, such as osteoporosis.

Animals↗
PubMed2003

A simple affinity spin tube filter method for removing high-abundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis.

Although it is possible to identify new proteins from crude cell extracts using proteomics technology, it is often difficult to elucidate low-abundant biomarkers in the presence of a large amount of high-abundant proteins in serum. We have developed a simple and rapid method using an affinity spin tube filter to remove high-abundant common proteins and enrich the low-abundant biomarkers. The affinity spin tube filter contains protein G, coupled with antibodies against either high-abundant proteins or specific proteins of interest. After incubating with serum, the flow-through or the elute was collected and analyzed by two-dimensional gel electrophoresis. By using this affinity spin tube filter, the possibilities of identifying new biomarkers are shown. This technique could be used for large-scale sample preparation for high-throughput proteomic analysis.

Biomarkers↗
PubMed2005

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Antibodies↗
PubMed2003

An improved method for the in vitro evolution of aptamers and applications in protein detection and purification.

One of the key components of proteomics initiatives is the production of high affinity ligands or probes that specifically recognize protein targets in assays that detect and capture proteins of interest. Particularly versatile probes with tremendous potential for use as affinity molecules are aptamers. Aptamers are short single-stranded DNA or RNA sequences that are selected in vitro based on affinity for a target molecule. Aptamers offer advantages over traditional antibody-based affinity molecules in their ease of production, regeneration and stability, largely due to the chemical properties of nucleic acids versus amino acids. We describe an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. We demonstrate the ability of aptamers that recognize thyroid transcription factor 1 (TTF1) to bind their target protein with high affinity and specificity, and detail their uses in a number of assays. The TTF1 aptamers were characterized using surface plasmon resonance, and shown to be useful for enzyme-linked assays, western blots and affinity purification.

Animals↗
PubMed2005

Proteomic scan for tyrosinase peptide antigenic pattern in vitiligo and melanoma: role of sequence similarity and HLA-DR1 affinity.

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95-104FMGFNCGNCK; aa175-182 LFVWMHYY; aa176-190FVWMHYYVSMDALLG; aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95-104FMGFNCGNCK, aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC, but did not react with the aa175-182LFVWMHYY and aa176-190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.

Alleles↗
PubMed2003

Mass spectrometry-based proteomics.

Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.

Animals↗
PubMedDate not supplied

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

Amino Acid Sequence↗
PubMed2006

Liquid chromatography with tandem mass spectrometry-based proteomic discovery in aging and Alzheimer's disease.

Systems biology offers enormous potential to understand the complexity of human brain aging and neurodegenerative diseases. Proteomics has an important role in these investigations because of its unique strengths and because of the potential central pathogenic contribution of pathological protein to several of these diseases. Here we have reviewed the methods and presented some examples of liquid chromatography-electrospray ionization-tandem mass spectrometry-based proteomics, with and without quantification using isotope-coded affinity tags, in the investigation of aging and Alzheimer's disease. As protocols and methods for improved quantitative high-throughput proteomics constantly improve, this approach will likely continue to provide deeper insight into human brain aging and neurodegenerative diseases.

Aging↗
PubMedDate not supplied

Proteomics technology and therapeutics.

1. Proteins are the workhorses of biological systems and, thus, it is not surprising that the field of proteomics has flourished in recent years. The present review briefly surveys the general proteomic approaches taken to identify drug targets and to evaluate drug efficacy and toxicity in the preclinical and clinical settings. These generally fall into three basic categories: a profiling approach, a functional approach and a structural approach. 2. The current 'tools' used and applied to the drug-discovery process will be highlighted. These include the two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, isotope-coded affinity tag and protein biochips. 3. Clearly, proteomics has begun to set a foothold in every stage of the drug-discovery process. Future developments in this area will likely make a significant impact in our quest for better, safer and cost-effective drugs.

Animals↗
PubMed2002

Quantitative protein profiling using two-dimensional gel electrophoresis, isotope-coded affinity tag labeling, and mass spectrometry.

Quantitative protein profiling is an essential part of proteomics and requires new technologies that accurately, reproducibly, and comprehensively identify and quantify the proteins contained in biological samples. We describe a new strategy for quantitative protein profiling that is based on the separation of proteins labeled with isotope-coded affinity tag reagents by two-dimensional gel electrophoresis and their identification and quantification by mass spectrometry. The method is based on the observation that proteins labeled with isotopically different isotope-coded affinity tag reagents precisely co-migrate during two-dimensional gel electrophoresis and that therefore two or more isotopically encoded samples can be separated concurrently in the same gel. By analyzing changes in the proteome of yeast (Saccharomyces cerevisiae) induced by a metabolic shift we show that this simple method accurately quantifies changes in protein abundance even in cases in which multiple proteins migrate to the same gel coordinates. The method is particularly useful for the quantitative analysis and structural characterization of differentially processed or post-translationally modified forms of a protein and is therefore expected to find wide application in proteomics research.

Affinity Labels↗
PubMed2006

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria.

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S-transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1>hGSTA2>hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.

Adult↗
PubMed2006

Lock-and-key motif as a concept for designing affinity adsorbents for protein purification.

The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.

Amino Acid Motifs↗
PubMed2004

A human proteome project with a beginning and an end.

Research activities centered on the ensemble of and individual human proteins have taken on numerous guises, some of which fall under the banner of what could be defined as a Human Proteome Project (HPP). However, the latter has yet to take-on the apparent global focus of its predecessor, the Human Genome Project. The reasons for this are both financial and technical. The disparate properties afforded to each protein by a 20-letter code render a single unifying approach difficult to implement, while the current limit of analytical detection has yet to deliver an entire proteome for even the simplest of microbes. The situation is complicated further by the fact that low abundance proteins dominate within any living cell. Thus, enhancement of signal-to-noise ratio by affinity ligands becomes of paramount importance if whole-organism proteomics is to be realized. The generation of such ligands (molecules exhibiting desirable affinity and selectivity for target) could provide the necessary focus and a task list with a definable beginning and end. Such a finite task list is considered essential if an HPP might one day deliver global coverage on a scale seen currently for the total DNA sequence of some 200 living organisms.

Human Genome Project↗
PubMed2006

New insights into molecular mechanisms of phagocytosis in Entamoeba histolytica by proteomic analysis.

The protozoan parasite Entamoeba histolytica ingests microorganisms and mammalian cells. Phagocytosis is essential for cell growth and is implicated in pathogenesis of E. histolytica. Phagocytosis consists of a number of steps including recognition of and binding to ligands on the target cells via a galactose/N-acetylgalactosamine-specific lectin, activation of a signaling pathway leading to cytoskeletal reorganization, and vesicle trafficking, all of which play distinct but coordinated roles in phagocytosis. Recent studies of proteomic analysis of purified phagosomes or affinity-purified Gal/GalNAc-binding proteins using reversed phase capillary liquid chromatography and ion trap tandem mass spectrometry enabled high throughput identification of proteins involved in phagosome biogenesis. These studies provided a list of proteins involved in the pathway and also shed light on the dynamic process of phagosome maturation. These approaches should provide significant insights into molecular mechanisms of phagosome biogenesis and help to elucidate the pathogenesis of this important parasite.

Animals↗
PubMed2002

Proteomic profiling of mechanistically distinct enzyme classes using a common chemotype.

Proteomics research requires methods to characterize the expression and function of proteins in complex mixtures. Toward this end, chemical probes that incorporate known affinity labeling agents have facilitated the activity-based profiling of certain enzyme families. To accelerate the discovery of proteomics probes for enzyme classes lacking cognate affinity labels, we describe here a combinatorial strategy. Members of a probe library bearing a sulfonate ester chemotype were screened against complex proteomes for activity-dependent protein reactivity, resulting in the labeling of at least six mechanistically distinct enzyme classes. Surprisingly, none of these enzymes represented targets of previously described proteomics probes. The sulfonate library was used to identify an omega-class glutathione S-transferase whose activity was upregulated in invasive human breast cancer lines. These results indicate that activity-based probes compatible with whole-proteome analysis can be developed for numerous enzyme classes and applied to identify enzymes associated with discrete pathological states.

Affinity Labels↗