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 865 records · Page 48Linked to original sources
PubMed2004

Proteomic analysis of DC-SIGN on dendritic cells detects tetramers required for ligand binding but no association with CD4.

DC-SIGN (dendritic cell specific intracellular adhesion molecule 3 grabbing non-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infection in trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV.

Amino Acid Sequence↗
PubMed2000

Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides.

A rapid means of identifying many components in an enriched mixture of proteins is enzymatic digestion of the entire protein fraction. This complex peptide mixture is then subjected to reversed-phase high performance liquid chromatography (HPLC) coupled on-line with a mass spectrometer capable of data-dependent ion selection for fragmentation (LC-tandem mass spectrometry; MS/MS). Thus, as many peptides as possible in the sample are fragmented to produce MS/MS spectra, which can then be searched against sequence databases. Ideally, one peptide from each protein in the mixture would be fragmented and identified. To this end, we employed an affinity selection method to capture cysteinyl peptides and thereby simplify the mixture. Both the captured cysteinyl and the noncysteinyl peptides are analyzed by LC-MS/MS, to increase the number of proteins identified. The method was tested on a limited set of standard proteins and applied to the analysis of a protein fraction obtained from isolated mitochondria treated with atractyloside. To further increase the number of different precursor ions selected for fragmentation, dynamic exclusion and ion selection from multiple narrow mass ranges of consecutive runs were employed.

Animals↗
PubMed2007

Carbonylation of adipose proteins in obesity and insulin resistance: identification of adipocyte fatty acid-binding protein as a cellular target of 4-hydroxynonenal.

Obesity is a state of mild inflammation correlated with increased oxidative stress. In general, pro-oxidative conditions lead to production of reactive aldehydes such as trans-4-hydroxy-2-nonenal (4-HNE) and trans-4-oxo-2-nonenal implicated in the development of a variety of metabolic diseases. To investigate protein modification by 4-HNE as a consequence of obesity and its potential relationship to the development of insulin resistance, proteomics technologies were utilized to identify aldehyde-modified proteins in adipose tissue. Adipose proteins from lean insulin-sensitive and obese insulin-resistant C57Bl/6J mice were incubated with biotin hydrazide and detected using horseradish peroxidase-conjugated streptavidin. High carbohydrate, high fat feeding of mice resulted in a approximately 2-3-fold increase in total adipose protein carbonylation. Consistent with an increase in oxidative stress in obesity, the abundance of glutathione S-transferase A4 (GSTA4), a key enzyme responsible for metabolizing 4-HNE, was decreased approximately 3-4-fold in adipose tissue of obese mice. To identify specific carbonylated proteins, biotin hydrazide-modified adipose proteins from obese mice were captured using avidin-Sepharose affinity chromatography, proteolytically digested, and subjected to LC-ESI MS/MS. Interestingly enzymes involved in cellular stress response, lipotoxicity, and insulin signaling such as glutathione S-transferase M1, peroxiredoxin 1, glutathione peroxidase 1, eukaryotic elongation factor 1alpha-1 (eEF1alpha1), and filamin A were identified. The adipocyte fatty acid-binding protein, a protein implicated in the regulation of insulin resistance, was found to be carbonylated in vivo with 4-HNE. In vitro modification of adipocyte fatty acid-binding protein with 4-HNE was mapped to Cys-117, occurred equivalently using either the R or S enantiomer of 4-HNE, and reduced the affinity of the protein for fatty acids approximately 10-fold. These results indicate that obesity is accompanied by an increase in the carbonylation of a number of adipose-regulatory proteins that may serve as a mechanistic link between increased oxidative stress and the development of insulin resistance.

Adipose Tissue↗
PubMedDate not supplied

Poly(glycidyl methacrylate/divinylbenzene)-IDA-FeIII in phosphoproteomics.

The study of protein phosphorylation has grown exponentially in recent years, as it became evident that important cellular functions are regulated by phosphorylation and dephosphorylation of proteins on serine, threonine and tyrosine residues. The use of immobilized metal affinity chromatography (IMAC) to enrich phosphopeptides from peptide mixtures has been shown to be useful especially prior to mass spectrometric analysis. For the selective enrichment applying solid-phase extraction (SPE) of phosphorylated peptides, we introduce poly(glycidyl methacrylate/divinylbenzene) (GMD) derivatized with imino-diacetic acid (IDA) and bound Fe(III) as a material. GMD is rapidly synthesized and the resulting free epoxy groups enable an easy access to further derivatization with, e.g., IDA. Electron microscopy showed that the synthesized GMD-IDA-Fe(III) for SPE has irregular agglomerates of spherical particles. Inductively coupled plasma (ICP) analysis resulted in a metal capacity of Fe(III) being 25.4 micromol/mL. To enable on-line preconcentration and desalting in one single step, GMD-IDA-Fe(III) and Silica C18 were united in one cartridge. Methyl esterification (ME) of free carboxyl groups was carried out to prevent binding of nonphosphorylated peptides to the IMAC function. The recovery for a standard phosphopeptide using this SPE method was determined to be 92%. The suitability of the established system for the selective enrichment and analysis of model proteins phosphorylated at different amino acid residues was evaluated stepwise. After successful enrichment of beta-casein deriving phosphopeptides, the established system was extended to the analysis of in vitro phosphorylated proteins, e.g. deriving from glutathione-S-transferase tagged extracellular signal regulated kinase 2 (GST-ERK2).

Amino Acid Sequence↗
PubMed2005

Comparative interactomics analysis of protein family interaction networks using PSIMAP (protein structural interactome map).

MOTIVATION: Many genomes have been completely sequenced. However, detecting and analyzing their protein-protein interactions by experimental methods such as co-immunoprecipitation, tandem affinity purification and Y2H is not as fast as genome sequencing. Therefore, a computational prediction method based on the known protein structural interactions will be useful to analyze large-scale protein-protein interaction rules within and among complete genomes. RESULTS: We confirmed that all the predicted protein family interactomes (the full set of protein family interactions within a proteome) of 146 species are scale-free networks, and they share a small core network comprising 36 protein families related to indispensable cellular functions. We found two fundamental differences among prokaryotic and eukaryotic interactomes: (1) eukarya had significantly more hub families than archaea and bacteria and (2) certain special hub families determined the topology of the eukaryotic interactomes. Our comparative analysis suggests that a very small number of expansive protein families led to the evolution of interactomes and seemed to have played a key role in species diversification. SUPPLEMENTARY INFORMATION: http://interactomics.org.

Algorithms↗
PubMed2004

Identification by a differential proteomic approach of heat shock protein 27 as a potential marker of atherosclerosis.

BACKGROUND: We hypothesized that normal and pathological vessel walls display a differential pattern of secreted proteins. We have recently set up the conditions for comparing secretomes from carotid atherosclerotic plaques and control arteries using a proteomic approach to assess whether differentially secreted proteins could represent markers for atherosclerosis. METHODS AND RESULTS: Normal endartery segments and different regions of endarterectomy pieces (noncomplicated/complicated plaques) were incubated in protein-free medium, and the released proteins were analyzed by 2D electrophoresis (2-DE). Among the differently secreted proteins, we have identified heat shock protein-27 (HSP27). Surprisingly, compared with control arteries, HSP27 release was drastically decreased in atherosclerotic plaques and barely detectable in complicated plaque supernatants. HSP27 was expressed primarily by intact vascular cells of normal arteries and carotid plaques (immunohistochemistry). Plasma detection of soluble HSP27 showed that circulating HSP27 levels are significantly decreased in the blood of patients with carotid stenosis relative to healthy subjects (0.19 [0.1 to 1.95] versus 83 [71.8 to 87.8]) ng/mL, P<0.0001). CONCLUSIONS: HSP27 secretion is decreased in complicated atherosclerotic plaques, and sHSP27 plasma levels are decreased in atherosclerotic patients compared with healthy subjects. Plasma sHSP27 levels could be a potential index of atherosclerosis, although further validation is needed in large patient cohorts.

Aged↗
PubMed2003

A novel approach toward development of a rapid blood test for breast cancer.

Mammography remains the diagnostic test of choice for breast cancer, but 20% of cancers still go undetected. Many serum biomarkers have been reported for breast cancer but none have proven to represent effective diagnostic strategies. ProteinChip mass spectrometry is an innovative technology that searches the proteome for differentially expressed proteins, allowing for the creation of a panel or profile of biomarkers. The objective of this study was to construct unique cancer-associated serum profiles that, combined with a classification algorithm, would enhance the detection of breast cancer Pretreatment serum samples from 134 female patients (45 with cancer, 42 with benign disease, 47 normal) were procured prospectively following institutional review board-approved protocols. Proteins were denatured, applied onto ProteinChip affinity surfaces, and subjected to surface enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry. The SELDI output was analyzed using Biomarker Pattern Software to develop a classification tree based on group-specific protein profiles. The cross-validation analysis of cancer versus normal revealed sensitivity and specificity rates of 80% and 79%, and for cancer versus benign disease, 78% and 83%, respectively. When 2 different chip surfaces were combined the sensitivity and specificity increased to 90% and 93%, respectively. The sensitivity and specificity of this technique are comparable to those of mammography and, if confirmed in a larger study, this technique could provide the means toward development of a simple blood test to aid in the early detection of breast cancer. The combination of SELDI ProteinChip mass spectrometry and a classification- and regression-tree algorithm has the potential to use serum protein expression profiles for detection and diagnosis of breast cancer.

Adult↗
PubMed2006

BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix.

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.

Alkaline Phosphatase↗
PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗
PubMed2005

A proteomic study of the apolipoproteins in LDL subclasses in patients with the metabolic syndrome and type 2 diabetes.

The exchangeable apolipoproteins present in small, dense LDL (sdLDL) and large, buoyant LDL subclasses were evaluated with a quantitative proteomic approach in patients with the metabolic syndrome and with type 2 diabetes, both with subclinical atherosclerosis and the B LDL phenotype. The analyses included surface-enhanced laser adsorption/ionization, time-of-flight mass spectrometry, and subsequent identification by mass spectrometry or immunoblotting and were carried out in LDL subclasses isolated by ultracentrifugation in deuterium oxide gradients with near physiological salt concentrations. The sdLDLs of both types of patients were enriched in apolipoprotein C-III (apoC-III) and were depleted of apoC-I, apoA-I, and apoE compared with matched healthy controls with the A phenotype. The LDL complexes formed in serum from patients with diabetes with the arterial proteoglycan (PG) versican were also enriched in apoC-III. In addition, there was a significant correlation between the apoC-III content in sdLDL in patients and the apparent affinity of their LDLs for arterial versican. The unique distribution of exchangeable apolipoproteins in the sdLDLs of the patients studied, especially high apoC-III, coupled with the augmented affinity with arterial PGs, may contribute to the strong association of the dyslipidemia of insulin resistance with increased risk for cardiovascular disease.

Animals↗
PubMed2005

A quantitative proteomic analysis of growth factor-induced compositional changes in lipid rafts of human smooth muscle cells.

Signals that promote proliferation and migration of smooth muscle cells (SMC) have been implicated in pathologic growth of hollow organs. Members of the platelet-derived growth factor (PDGF) family, potent mitogens and motility factors for SMC, have been shown to signal through cholesterol-enriched lipid rafts. We recently demonstrated that PDGF-stimulated DNA synthesis in urinary tract SMC was dependent on the integrity of lipid rafts. Despite its known ability to rapidly alter discrete proteins within rafts, the effect of PDGF on overall raft protein composition is unknown. In this study, we employed isotope coded affinity tag (ICAT) analysis to evaluate PDGF-induced protein changes in lipid rafts of primary culture human SMC. Following acute (i.e., 15 min) exposure of SMC to PDGF, 23 proteins increased in rafts >20%. In contrast, raft localization of only three proteins increased after 12 h of PDGF treatment. Among the proteins that increased at 15 min were the glycophosphatidylinositol-anchored proteins Thy-1, 5'-nucleotidase, and CD55, the cytoskeletal proteins actin, actinin, tropomyosin-3 and -4, and the endocytosis-related proteins clathrin and beta-adaptin. In addition, eight Rho family members were localized to rafts by ICAT analysis. Collectively, these observations suggest a role for lipid rafts in regulation of PDGF-stimulated changes in the cytoskeleton.

Cells, Cultured↗
PubMed2005

Reduction of the concentration difference of proteins in biological liquids using a library of combinatorial ligands.

The discovery of polypeptides and proteins with relevance to a particular biological state is complicated by their vast number and concentration range in most biological mixtures. Depletion methodologies are frequently used to remove the most abundant species; however, this removal not only fails significantly to enrich trace proteins, it may also nonspecifically deplete them due to their interactions with the removed high-abundance proteins. Here we report a simple-to-use methodology that reduces the protein concentration range of a complex mixture like whole serum through the simultaneous dilution of high-abundance proteins and the concentration of low-abundance proteins. This methodology utilizes solid-phase ligand libraries of immense diversity, generated by "split, couple, recombine" combinatorial chemistry, that are used for affinity-based binding to the proteins of a given mixture. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that many peptides or proteins that are undetectable by classical analytical methods become easily accessible. The reduction in the dynamic range of unfractionated serum is specifically described along with treatment of other proteomes such as extracts from Escherichia coli, chicken egg white and cell culture supernatant. Mono- and bi-dimensional electrophoresis (1-DE and 2-DE respectively) and surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-TOF-MS) technology demonstrate the reduction in protein concentration range. Combining this approach with additional fractionation methods further increased the number of detectable species.

Adsorption↗
PubMed2006

Mass spectrometry-based immunoassays for the next phase of clinical applications.

Recent applications of affinity mass spectrometry into clinical laboratories brought a renewed interest in immunoaffinity mass spectrometry as a more specific affinity method capable of selectively targeting and studying protein biomarkers. In mass spectrometry-based immunoassays, proteins are affinity retrieved from biological samples via surface-immobilized antibodies, and are then detected via mass spectrometric analysis. The assays benefit from dual specificity, which is brought about by the affinity of the antibody and the protein mass readout. The mass spectrometry aspect of the assays enables single-step detection of protein isoforms and their individual quantification. This review offers a comprehensive review of mass spectrometry-based immunoassays, from historical perspectives in the development of the immunoaffinity mass spectrometry, to current applications of the assays in clinical and population proteomic endeavors. Described in more detail are two types of mass spectrometry-based immunoassays, one of which incorporates surface plasmon resonance detection for protein quantification. All mass spectrometry-based immunoassays offer high-throughput targeted protein investigation, with clear implications in clinical research, encompassing biomarker discovery and validation, and in diagnostic settings as the next-generation immunoassays.

Animals↗
PubMed2004

Membrane receptor mapping: the membrane topography of Fc(epsilon)RI signaling.

Ligand binding to membrane receptors initiates cascades of biochemical events leading to physiological responses. Hundreds of proteins and lipids are implicated in signaling networks and programs in genomics and proteomics are continuously adding new components to the signaling "parts lists". Here, we generate high resolution maps of signaling networks using cytoplasmic face-up membrane sheets that can be labeled with immunogold probes (3-10 nm) and imaged in the transmission electron microscope. Our model system is the mast cell and we focus on mapping the topography of the high affinity IgE receptor, Fc(epsilon)RI, its associated tyrosine kinases, Lyn and Syk, and the signaling proteins that propagate signals from these kinases. Crosslinked receptors and their signaling partners segregate during signaling to multiple, dynamic membrane domains, including a transient Fc(epsilon)RI-Lyn domain and at least two other distinct domains, one characterized by the presence of receptor, Syk and multiple signaling proteins, but not Lyn (primary signaling domains), and one characterized by the presence of LAT and PLCgamma1 but not receptor (secondary signaling domains). PI 3-kinase associates with both primary and secondary signaling domains and may help to recruit specific signaling proteins through the local remodeling of inositol phospholipids. The lipid raft markers, GM1 and Thy-1, fail to localize in native membrane sheets either with each other or with signaling domains. We introduce new probes to localize multiple signaling molecules on the same membrane sheet and new computational tools to capture and analyze their topographical relationships. In the future, we expect that high resolution maps of signaling networks will be integrated with chemical kinetic analyses, with cell fractionation data and with a range of real-time fluorescence measurements, into mathematical models with power to predict mechanisms that regulate the efficiency, specificity, amplitude and duration of signaling pathways.

Animals↗
PubMed2004

[The tortuose path of human evolution].

The human genome sequencing and also the chimpanzee's, among others alive beings in every kingdom, and the similarities between genomes, due to an unique life origin, has developed two movements in the Biological Sciences to the XXI Century. In the first place to find a genetic explanation to understand the genomic affinity, very narrow in many cases, in front of the reality, with abyssal differences in behavior, which undervalue the significance of the genomic resemblance between the alive beings. The second movement is directed to ethological studies of the animal behavior and conduct, as a real fact. Special interest has the comparative studies since the two points of view, the genome and proteome in one side and ethology in front, both related to the Homo sapiens sapiens and his relatives, the great anthropomorphic apes, and consequently beside the names of Craig Venter, Collins and collaborators, artificers of the human genome sequencing, appear, unde revaluation, those of Leakey, Goodall, Fossey and Galdikas, specialists in anthropology and ethology of chimpanzees, gorillas and orangutans. These ideas are complementary in order to a better understanding of the origin, human evolution and behavior, shedding light on human cognitive abilities.

Animals↗
PubMed2004

Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis.

SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, IkappaB, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the C-terminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases, in a manner similar to ubiquitinylation. Identification of SUMO-regulated proteins is hampered by the fact that many sumoylated proteins are present at a level below normal detection limit. This limitation was overcome by either in vivo overexpression of Myc-SUMO or in vitro sumoylation with excess biotin-SUMO and Ubc9. Sumoylated proteins so obtained were affinity purified or isolated by immunoprecipitation. The isolated sumoylated proteins were identified by sequence analysis using mass spectrometric methods. Results reveal that several heterogeneous nuclear ribonucleoproteins (hnRNPs), zinc finger proteins, and nuclear pore complex proteins were sumoylated. The sumoylation of hnRNP A1, hnRNP F, and hnRNP K were confirmed in vivo by coimmunoprecipitation. In view of the facts that hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our findings suggest that sumoylation could play an important role in regulating mRNA metabolism.

Amino Acid Sequence↗
PubMedDate not supplied

Current and emerging commercial optical biosensors.

The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis.

Biosensing Techniques↗
PubMed2005

High-throughput solubility assay for purified recombinant protein immunogens.

A high-throughput assay is described for analysis of the solubility of purified recombinant proteins. The assay is based on affinity purification of proteins in the presence of chaotropic agents followed by a dilution and incubation step to investigate the solubility in the absence of high concentrations of such agents. The assay can be performed in a 96-well format, which makes it well suited for high-throughput applications. For 125 recombinant proteins expressed as part of an antibody-based proteomics effort, experimental solubility data were compared to calculated hydrophobicity values based on the amino acid sequence of each protein. This comparison showed only weak correlation between the theoretical and experimental values, which emphasizes the importance of experimental assays to determine the solubility of recombinant proteins.

Antibodies↗