Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography

Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor’s cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC–MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC–MS/MS.     

Proteomic analysis of human blood serum using peptide library beads

Human serum is thought to contain key information for diagnostics of human disease. However, no single technology is currently nor might ever be available to cope with the complexity and dynamic range of the serum proteome. We here report a large-scale proteomic study of human blood serum using peptide library beads and mass spectrometry. Serum proteins are adsorbed onto polymeric beads coated with a combinatorial library composed of millions of hexameric peptide baits. Analysis of the eluates from this combinatorial library (as obtained with 3 eluants of different strength, able to release 99% of the retentate) via liquid chromatography coupled to high-resolution mass spectrometry resulted in the identification of 1559 proteins or 3869 proteins, respectively, depending on how 95% confidence was estimated. In either case, the analysis showed that ligand beads are able to capture a large number of proteins in a single operation. The ligand bead bound fraction appeared to have a lower dynamic range when compared to the starting material, due to a "normalization" of the protein concentrations in the original mixture. We find that extensive information on the protein composition of complex samples such as serum can be obtained using ligand beads and that these beads enrich the proteomic tool box.     

Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin

Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.     

Proteomics analysis of human brain tissue infected by street rabies virus

In order to extend the knowledge of rabies pathogenesis, a two-dimensional electrophoresis/mass spectrometry based postmortem comparative proteomics analysis was carried out on human brain samples. Alteration in expression profile of several proteins was detected. Proteins related to cytoskeleton, metabolism, proteasome and immune regulatory systems showed the most changes in expression levels. Among these groups, the cytoskeleton related proteins (dynein light chain, β-centractin, tubulin alpha-1C chain and destrin) and metabolism associated proteins (fatty acid-binding protein, macrophage migration inhibitory factor, glutamine synthetase and alpha enolase) were the main altered proteins. These alterations may be considered as an evidence of disturbances in neuronal key processes including axonal transport, synaptic activity, signaling and metabolic pathways in rabies virus infected human brain.     

OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL

The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads.     

Analysis and isolation of human transferrin receptor using the OKT-9 monoclonal antibody covalently crosslinked to magnetic beads.

A method is described for the use of magnetic beads as a solid phase for the immunoprecipitation of labeled proteins. The anti-human transferrin receptor monoclonal antibody OKT-9 has been coupled to sheep anti-mouse IgG1-coated magnetic beads using the crosslinking agent dimethyl pimelimidate. The transferrin receptor is readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography following immunoprecipitation from 35S-labeled cell lysates. When compared with precipitations using OKT-9 coupled to protein G Sepharose the magnetic beads result in fewer nonspecific bands. The protocol described is generally applicable to the identification of labeled proteins. In addition, because magnetic beads are amenable to covalent crosslinking procedures they can be used for the purification of proteins from complex mixtures. Covalently crosslinked OKT-9 sheep anti-mouse IgG1-coated magnetic beads have been used to affinity purify unlabeled transferrin receptor from cell lysates giving comparable purity and yield to transferrin Sepharose isolated transferrin receptor. The major advantages offered by magnetic beads compared to conventional affinity matrices are low nonspecific binding and the rapidity with which the purification can be performed.     

Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.     

A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO₄ and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18–oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity.     

Profiling of the CD4 receptor complex proteins

Intermolecular complexes produced by the CD4 molecule were studied. To preserve the integrity of weak protein-protein interactions of the CD4 antigen, cells were lysed in a mild nonionic detergent Brij97. Protein constituents of the complex were identified by our previously proposed fluorescence immunoprecipitation assay with subsequent mass spectrometry. In total, 26 proteins associated with CD4 were identified on CEM cells. The CD4 complex included the following major components: tyrosine phosphatase CD45, transferrin receptor CD71, tyrosine kinase Lck, and a lymphocyte phosphatase-associated phosphoprotein LPAP. The CD4 complex also contained some components of cytoskeleton and heat shock proteins. The association between CD4, CD71, and CD45 molecules was confirmed by immunoblotting. The CD4 complexes were not detected on the U937 myeloid cells lacking Lck and LPAP. We attempted to quantitatively characterize the CD4 complex composition.     

Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.     

A controlled pore glass bead assay for the measurement of cytoplasmic and nuclear glucocorticoid receptors

An assay for the quantitation of cytoplasmic and nuclear glucocorticoid receptors in lymphoid tissue has been developed using controlled pore glass (CPG) beads. Soluble receptor--3H-steroid complex (cytosol or nuclear extract) is adsorbed quantitatively within the crevasses of porous glass beads. Excess labeled steroid as well as most non-specifically bound steroid is easily washed away, leaving the hormone-receptor complex retained by the beads. Bound 3H-steroid is eluted with ethanol and measured for radioactivity. This procedure which is simple, rapid, and highly reproducible is carried out using frozen samples (stable for many months) containing as few as 1 X 10(7) cells. A comparison of the CPG assay to dextran coated charcoal and a whole cell assay demonstrates that CPG and dextran coated charcoal give equivalent measurements of cytosolic receptor concentration, while the CPG and whole cell assays provide equivalent values for total receptor content.     

Proteolytic signaling by TNFalpha: caspase activation and IkappaB degradation

Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IkappaBalpha. Caspases are activated using mechanisms of oligomeration and 'self-controlled proteolysis'. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-kappaB is dependent on the degradation of IkappaBalpha. IkappaB kinase (IKK) phosphorylates IkappaBalpha at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-kappaB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-kappaB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-kappaB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die.     

Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles

Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.     

Proteomic analysis of Gal/GalNAc lectin-associated proteins in Entamoeba histolytica

Contact-dependent killing and phagocytosis of target cells by Entamoeba histolytica trophozoites is mediated by the galactose (Gal) and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. Previous work has suggested that this lectin functions as part of a signal transduction complex. To identify proteins that might be part of this complex, amebic trophozoites were bound to GalNAc-BSA-labeled magnetic beads and lysed. Bound proteins were eluted from the beads and analyzed by tandem mass spectrometry. Along with the Gal/GalNAc lectin subunits, several cytoskeletal proteins, potential signaling proteins, and a novel transmembrane protein, consistently purified with the GalNAc-BSA beads.     

Immunological detection of adulteration of ground meats by meats of other origins

Specific and sensitive assays for immunoglobulin G in pork, chicken, and beef were developed using polyester cloths coated with respective anti-immunoglobulin G antibodies. The captured immunoglobulin G proteins were detected by their reactions with the respective IgG antibody horseradish peroxidase conjugates and a chromogenic peroxidase substrate. This sensitive assay takes only 30 minutes and can detect the adulteration of ground meats.     

Detection of human blood by cloth-based enzyme immunoassay of immunoglobulin G

A specific and sensitive assay for the detection of human blood was developed using polyester cloth coated with goat anti-human IgG antibody to capture human IgG, an abundant and stable protein in blood. The captured IgG was detected by the reaction between goat anti-human IgG antibody-peroxidase conjugate and a chromogenic peroxidase substrate. Because the assay is simple and rapid, and permits simultaneous analysis of multiple samples, it has the potential to be used as a forensic test for human blood.     

Immunoaffinity-based mass spectrometric characterization of immunoreactive proteins of Salmonella Typhi

Salmonella Typhi, a human-restricted Gram negative enterobacteriaceae, is the causative agent of typhoid fever in human being. The available serodiagnostic tools for the diagnosis of typhoid fever lack sensitivity and/or specificity. This study aimed to identify the immunoreactive proteins of S. Typhi that could help to develop improved diagnostic tools. Here, we performed immunoaffinity-based proteomic approach that uses charged columns to retrieve IgG and IgM antibodies from the plasma of typhoid patients followed by capture of S. Typhi proteins. These proteins were then characterized by mass spectrometry and bioinformatics tools. Using this approach, we identified 28 immunoreactive proteins of S. Typhi, in which 14 proteins were captured by IgG charged column and 4 proteins were captured by IgM column. We also identified 10 proteins (hlyE, rfbH, dapD, argI, glyA, pflB, trxB, groEL, tufA and pepD) captured by both columns. The prediction of antigenicity and immunogenicity resulted that 22 proteins were antigenic while 6 were non-antigenic on the scale of 0.4 threshold value of VaxiJen. These proteins successfully simulated the immune system in silico and in response higher amount of antibodies‘ titers were recorded in C-IMMSIM, confirming the immunogenic nature of these proteins. The identified proteins are of diverse nature and functions including those involved in virulence and pathogenesis, energy metabolism, cell development, biosynthesis of amino acids, regulatory functions and biosynthesis of cofactors. The findings of this study would be helpful in the development of improved vaccines and diagnostic tools for typhoid fever.     

A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid in shellfish products

A lateral flow immunochromatographic (LFIC) test strip based on a colloidal gold-monoclonal antibody (McAb) conjugate was developed for on-site rapid detection of okadaic acid (OA) in shellfish. It applies a competitive format using an immobilized toxin conjugate and free toxin present in samples. The McAb against OA was conjugated with 20-nm colloidal gold as detector reagent. The toxin in the sample competed with the immobilized toxin to bind to the gold conjugated with McAb. The colloidal gold/McAb/toxin mobile complex was not captured by OA-bovine serum albumin (BSA) on the test line, but it was captured by goat anti-mouse immunoglobulin G (IgG) on the control line. The color density of the test line correlated with the concentration of toxin in the range of 10-50 ng ml(-1). The qualitative detection limit of 150 μg kg(-1) sample was close to the European Union (EU) regulatory limit (160 μg kg(-1)). Therefore, these strips were able to directly and qualitatively estimate the consuming safety of shellfish. They require no equipment because of available visual results, and they screened numerous samples within 10 min. The results were further confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). As a food safety screening tool, the test strips are convenient and useful to rapidly on-site test the presence of OA in shellfish products.     

Single-bead-based immunofluorescence assay for snake venom detection

In this communication, we reported a rapid and sensitive immunofluorescence method for the detection of snake venom by using microscale polystyrene beads as platform combined with semiconductor quantum dots (Qdots) as fluorescence label. Briefly, control rabbit IgG or capture antibody for venom was covalently immobilized onto the microspheres (surface activated with carboxyl group, dyed with different color) to form the control or capture beads. When incubated with the testing samples, the venom binds to the specific capture beads to form the complex through antibody-antigen interaction. Then, the second antibody conjugated Qdot was added, which targeted the Qdot to bind to the capture bead/antigen complex. The complex can be directly observed under a UV microscope. The system was applied to the testing of Naja kaouthia venom. Fluorescent microscopic images of QD-labeled capture beads demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of capture beads, indicating that the conjugated antibody molecules remained active and were able to recognize their specific target in solution. The detection limit of this method was 5-10 ng/mL. The detection could be completed within 3 h.