Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.     

Determining protein kinase substrate specificity by parallel solution-phase assay of large numbers of peptide substrates

We describe here a protocol for determining the activity of protein kinases on a large set of peptide substrates. Biotin-tagged peptides are arrayed in multiwell plates and incubated in solution with the kinase of interest and radiolabeled ATP. Reactions are then spotted simultaneously onto a streptavidin membrane, which is washed, dried, and analyzed by autoradiography or phosphor imaging. Differences in the extent of radiolabel incorporation into the various peptide substrates provide a measure of the sequence specificity of the kinase. This approach is a faster, more sensitive, and more generally applicable method for determining kinase phosphorylation motifs than older peptide library screening approaches based on Edman sequencing. The procedure is readily adaptable to other applications that require parallel processing of many kinase reactions, such as screening for small molecule inhibitors. In the format described here, preparation of stock plates prior to running the reactions will require about 4 days. Afterwards, the protocol takes approximately 6 hours to perform.     

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting

We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTip(C18) microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTip(C18)-based purification of protein prior to MALDI-TOF-MS analysis.     

Chimeric synthetic peptide as antigen for immunodiagnosis of HIV-1 infection

One chimeric peptide incorporating antigenic sequences from the gp41 transmembrane region (peptide H-18) and the gp120 envelope region (peptide H-15) corresponding to amino acids (587-617) on gp41 and (495-516) on gp120 of human immunodeficiency virus (HIV 1) was synthesized. Both sequences were separated by two glycine residues. This peptide was evaluated as antigen in an ultramicro-enzyme-linked immunosorbent assay (UMELISA) with samples derived from HIV-1 (n = 30) with different titers of antibodies and healthy blood donors (n = 30). The results were compared to plates coated with monomeric peptides and to plates coated with two monomeric peptides together. Results demonstrated that monomeric peptides gp41 (H-18) and gp120 (H-15) were good as antigens with samples that present antibodies to these regions. The chimeric peptide was the most antigenic. Those results may be related to the peptide structure, adsorption to the solid surface, and epitope accessibility to the antibodies. This chimeric peptide would be very useful for HIV-1 diagnostics.     

Formation of chitin-based nanomaterials using a chitin-binding peptide selected by phage-display

Targeting polymers with peptides is an efficient strategy to functionalize biomaterials. Phage display technology is one of the most powerful techniques for selecting specific peptides for a wide variety of targets. A method to select a chitin-binding peptide from a 12-mer random peptide library was successfully performed against chitin immobilized in wells of microtiter plates. The synthetic chitin binding peptide (ChiBP) could bind to chitin beads and disrupt their structure. This selected peptide was successfully used to immobilize alkaline phosphatase on chitin. In addition, the peptide could induce colloidal chitin in water to form a chitin coat on the surface of plastic tubes. Scanning electron microscopy (SEM) revealed that the peptide could induce colloidal chitin and chitohexaose to form networks when the temperature was raised to 42°C.     

High-performance partition chromatography of peptides on a new cross-linked agarose

A new cross-linked agarose in bead form (20-40 microns in diameter) has been used for partition chromatography in two-phase solvent systems to give columns exhibiting efficiencies of several thousand theoretical plates. The efficiency of this high-performance partition column (70-80 plates per cm) far exceeds that of the most commonly used soft gel Sephadex G-50 (10 plates per cm). The column is operated under moderate pressure (5 p.s.i.) and can be regenerated. The new procedure appears capable of separating a peptide of moderate size, such as beta-endorphin (31 residues), from the majority of possible single-deletion sequences.     

A microplate assay specific for the enzyme aggrecanase

We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.     

Design of a synthetic adhesion protein by grafting RGD tailed cyclic peptides on bovine serum albumin

A synthetic adhesion protein was designed by chemical grafting of the RGD tailed cyclic peptide cyclo[-d-Val-Arg-Gly-Asp-Glu(-Ahx-Tyr-Cys-NH₂)-] on the carrier protein bovine serum albumin (BSA). The cyclic conformation of the RGD motif grafted on the protein mimics the conformation of the motif displayed in native adhesion proteins such as fibronectin. The adhesion of the cells on polystyrene coated with the conjugate BSA–peptide was similar or even better than the one obtained when the proadhesive protein fibronectin was coated on the plates. Results also indicated that covalent coupling of the peptide on BSA is not absolutely required, since simple adsorption of the peptide on the protein coated on plates was efficient for enhancing cell adhesion. These results show that polystyrene support can be reconditioned with conformationally constrained RGD peptides to enhance cell adhesion on solid supports. The same methodology can be adapted for the development of new biomaterials based on the recognition of specific peptides.     

Identification and Properties of Plateins,Major Proteins in the Cortical Alveolar Plates of Euplotes1

Morphogenesis of the ciliate cortex has been viewed as an attractive model system for studying the mechanisms behind the ordered assembly of subcellular structure. Based on the assumption that identifying protein components of the cortex would facilitate the study of cortical assembly, I have produced a number of monoclonal antibodies directed against components of the cortex of Euplotes aediculatus. Several of these antibodies react with the proteins comprising the alveolar plates. These thin polygonal scales, each enclosed within a flattened membranous sac (alveolus) just beneath the cell membrane, tightly abut in a confluent monolayer that appears to lend form and rigidity to the Euplotes cell cortex. Reactivity and specificity of these monoclonal antibodies for the alveolar plates was shown by immunofluorescence staining of whole-cell preparations and of cryosections, and by immuno-gold staining of thin sections by electron microscopy. On immunoblots of SDS-PAGE separated whole-cell extracts, the plate proteins are revealed as two to three closely spaced bands centered at an M_r of 97 kDa, and a larger relative at 125 kDa. Comparative peptide mapping reveals that the members of the 97-kDa protein cluster are closely related. However, the 125-kDa polypeptide varies significantly from the 97-kDa members, and hence is not likely a synthetic precursor. Because bands of these M_r values are prominent in Coomassie blue-stained gels of whole-cell extracts, and are greatly enriched in purified cortical preparations, they likely represent the major proteins comprising the alveolar plates of E. aediculatus. I have proposed the name platein for this family of proteins.     

Purified HLA class II peptide complexes can induce adherence and activation of peptide-specific human T cell clones.

An initial event in T cell activation is the specific adherence of T cells via their T cell receptor to the MHC peptide complex. We have studied this adherence by incubating T cells with preformed HLA DR4Dw4 peptide complexes attached to a solid support. Adherence of sodium 51Cr-labeled T cell clones specific for the influenza hemagglutinin peptide, HA 307-319, was maximal after 15 min and was specific for the HLA DR4Dw4-HA 307-319 complex. The binding was temperature dependent and could be blocked with azide or protein kinase C inhibitors, indicating that for adherence the T cells need to be metabolically active and have a functioning protein kinase C pathway. The adherence could be blocked with CD4- or CD3-reactive murine mAb, suggesting that the TCR and CD4 molecules work in concert to induce strong adherence to the HLA DR4Dw4-HA 307-319 complex. A subsequent event in T cell activation is proliferation, which is thought to need additional proteins such as IL-1 or other adhesion molecules. MHC peptide complexes coated on microtiter plates also induced proliferation in the human T cell clones. Removal of any monocytes by treatment of human T cell clones with anti-CD14 in conjunction with C, followed by purification over a nylon wool column, did not abrogate proliferation. After prolonged culture of the T cell clones in plates coated with peptide-pulsed HLA DR4Dw4 in the presence of IL-2, the T cell clones continued to proliferate in response to peptide. These results suggest that human T cell clones do not require a second signal from a monocyte or other APC to proliferate.     

High biological activity of a recombinant protein immobilized onto polystyrene

The adsorption characteristics of glutathione S-transferases (GST) genetically fused with polystyrene (PS)-binding peptides (PS-tags) on PS plates with increase in hydrophilicity were studied to clarify the mechanisms of the specific interaction between the PS-tag-fused protein and PS plates. GST fused with the PS-tag PS19 (RAFIASRRIKRP) preferentially interacted with hydrophilic PS plates, even in the presence of high concentrations of competitors such as Tween 20 and BSA. Both basic and aliphatic amino acids in the PS-tags were involved in the specific interaction of PS-tags with the surface of the hydrophilic PS plate. Genetic fusion of the PS19 variants, PS19-4 (RAIARRIRR) and PS19-6 (RIIIRRIRR), further improved the immobilization yield of GST in the presence of a high concentration of the competitor BSA (50 mg/mL). The PS19-6 peptide specifically interacted with the surfaces of various hydrophilic PS plates, especially in the presence of Tween 20. Higher remaining activity was detected on all of the hydrophilic PS plates immobilized with GST-PS19-6 in comparison with those with wild-type GST and GST-PS19, and the remaining activity was further increased by the addition of Tween 20 in the adsorption state. The PS19-6 peptide developed in this study is therefore very useful as an affinity tag that can immobilize a target protein directly onto various hydrophilic PS supports with high remaining activity.     

Parallel synthesis of peptide libraries using microwave irradiation

The application of microwave irradiation to solid-phase peptide synthesis increases product purity and reduces reaction time. Parallel synthesis in 96-well polypropylene filter plates with microwave irradiation is an efficient method for the rapid generation of combinatorial peptide libraries in sufficient purity to assay the products directly for biological activity without HPLC purification. In this protocol, the solid-phase support is arrayed into each well of a 96-well plate, reagents are delivered using a multichannel pipette and a microwave reactor is used to complete peptide coupling reactions in 6 min and Fmoc-removal reactions in 4 min under temperature-controlled conditions. The microwave-assisted parallel peptide synthesis protocol has been used to generate a library of difficult hexa-beta-peptides in 61% average initial purity (50% yield) and has been applied to the preparation of longer alpha- and beta-peptides. Using this protocol, a library of 96 different hexapeptides can be synthesized in 24 h (excluding characterization).     

Mineral oil-, glycerol-, and Vaseline-coated plates as matrix-assisted laser desorption/ionization sample supports for high-throughput peptide analysis

A novel protocol for rapid and high-quality sample preparation prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed by coating bare stainless steel plates with one of three adhesives: mineral oil, glycerol, or Vaseline. The advantages of these three adhesive coats are that they take little time to both prepare and wipe away, hold the matrices to prevent them from flying from the support, reduce the background matrix, and affect neither the resolution of the peptide peaks nor the accuracy of their determined molecular masses. Consequently, the signal intensity, detection limit, and tolerance of the analytes to contaminants on the three adhesive-coated plates are improved. In the two strategies of on-plate desalting and concentration of the peptide mixture, all three adhesives reduced the loss of peptides, especially in the case of larger molecular mass peptides. The microscope and stereomicroscope images of the deposited droplets showed that after dropping onto the adhesive coats, the droplets formed a reduced spot size, were more homogeneous, and showed sticky crystallization. Therefore, this is an easy-to-use, reproducible, highly sensitive, tolerant (to salts), and high-throughput method of peptide sample preparation for MALDI-TOF MS analysis.     

纤溶酶产生菌—藤黄微球菌的筛选、鉴定及纤溶酶基因的克隆

通过血粉培养基富集、纤维蛋白培养基筛选,从自然环境中分离到一株纤溶酶产生菌,命名为ML909.利用传统细菌分类鉴定方法对其形态和生理生化特征进行了研究,发现其与藤黄微球菌的特征相符,16S rDNA序列分析的结果也表明它与藤黄微球菌的16S rDNA序列的同源性高达99%,因此该茵在系统分类学上属于微球茵属(Micrococcus Cohn)藤黄微球菌(Micrococcus luteus).通过PCR方法对其纤溶酶的编码区序列进行了克隆和序列分析,基因登录GenBank,登录号为EU232121.     

Characterizing and optimizing protease/peptide inhibitor interactions, a new application for spot synthesis

A new method is presented that uses parallel peptide array synthesis on cellulose membranes to characterize protease/peptide inhibitor interactions. A peptide comprising P5-P4' of the third domain of turkey ovomucoid inhibitor was investigated for both binding to and inhibition of porcine pancreatic elastase. Binding was studied directly on the cellulose membrane, while inhibition was measured by an assay in microtiter plates with punched out peptide spots. The importance of each residue for binding or inhibition was determined by substitutional analyses, exchanging every original amino acid with all other 19 coded amino acids. Seven hundred eighty individual peptides were investigated for binding behavior to porcine pancreatic elastase, and 320 individual peptides were measured in inhibition experiments. The results provide new insights into the interaction between the ovomucoid derived peptide and subsites in the active site of elastase. Combining these data with length analysis we designed new peptides in a step-wise fashion which in the end not only inhibited elastase 400 times more strongly than the original peptide, but are highly specific for the enzyme. In addition, the optimized inhibitor peptide was protected against exopeptidase attack by substituting D-amino acids at both termini.     

169 Polymorphic assembly of computationally designed hydrophobic-rich collagen peptides

We seek to understand how the position and length of hydrophobic content within a collagen peptide sequence dictates morphology of self-assembly. We modeled collagen assembly using diffusion limited aggregation¹ (DLA) (Parkinson et al. 1995). of discretized, rigid rods composed of hydrophilic and hydrophobic spheres. Simulations predicted that the inclusion of short hydrophobic domains should direct the assembly of lamellar structures. We designed a set of collagen peptide sequences with six, five and four contiguous nonpolar residues. Electron microscopy of aggregates revealed the peptide with six nonpolar residues self-assembled into uniform fibrils and the peptide with five residues assembled into both fibrils and plates, while including four hydrophobic residues that formed only plates. This polymorphic behavior can be explained by packing models of rod vs. screw-like-particles.     

Mitochondrial regulation of hypoxia-induced increase of adrenomedullin mRNA in HL-1 cells

Hypoxia upregulates the expression of the cardioprotective peptide adrenomedullin in cardiomyocytes. We characterized this pathway in murine HL-1 cardiomyocytes. Inhibition of mitochondrial complexes I, III, and IV largely, but not completely, reduced hypoxic adrenomedullin mRNA increase in gas-impermeable culture plates. Complex III inhibition was also effective in permeable culture plates, so that this effect is unlikely due to intracellular oxygen redistribution, whereas complex I blockade was ineffective in permeable plates. Complex II does not participate in this effect, as shown by chemical and siRNA inactivation. ROS scavenging by nitroblue tetrazolium and general flavoprotein inhibition by diphenyleniodonium nearly abrogated the hypoxic adrenomedullin mRNA increase. Thus, ROS production by flavoproteins is crucial for hypoxic upregulation of adrenomedullin mRNA in murine HL-1 cardiomyocytes. These ROS originate both from the mitochondrial complex III and from additional, presumably extramitochondrial, sources. Mitochondrial oxygen consumption appears to have impact on oxygen availability at these extramitochondrial sensors.     

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

Quantitative measurement of bitagged recombinant proteins using an immunometric assay: application to an anti-substance P recombinant antibody

We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer. In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb. A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference. The method was applied to the direct measurement, in periplasmic extracts, of the total and active fractions of an ScFv produced at different induction temperatures.