Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta2-microglobulin (beta2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients.     

Analysis of high molecular mass proteins larger than 150 kDa using cyanogen bromide cleavage and conventional 2-DE

Proteomic approaches including high-resolution 2-DE are providing the tools needed to discover disease-associated biomarkers in complex biological samples. Although 2-DE is an extremely powerful approach to analyze the proteome, the separation of proteins with extreme molecular masses still remains an issue requiring improvement. Because high molecular mass (HMM) proteins larger than 150 kDa have already been observed to be differentially expressed in several pathologies such as cancer, we developed an original strategy to analyze this part of the proteome that is not easily separated by 2-DE in polyacrylamide gels. This strategy is based on the 2-DE separation of cyanogen bromide (CNBr) fragments of purified HMM protein fractions, and combines techniques including SEC fractionation, TCA precipitation, CNBr cleavage, 2-DE and MS analysis. The method was first tested on a model protein, the BSA. Preliminary results obtained using colonic tissues led to the identification of six HMM proteins with M(r) comprised between 163 and 533 kDa in their reduced state. These results demonstrated that our CNBr/2-DE approach should provide a powerful tool for identification of new biomarkers larger than 150 kDa.     

Development of a homogeneous immunoassay system using protein A fusion fragmented Renilla luciferase

A homogeneous immunoassay system was developed using fragmented Renilla luciferase (Rluc). The B domain of protein A was fused to two Rluc fragments. When complexes between an antibody and fragmented Rluc fusion proteins bind to target molecules, the Rluc fragments come into close proximity and the luminescence activity of fragmented Rluc is restored by complementation. As proof-of-principle, this fragmented Rluc system was used to detect E. coli homogeneously using an anti-E. coli antibody.     

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.     

Cyanogen bromide cleavage generates fragments suitable for expressed protein and glycoprotein ligation

Herein cyanogen bromide is employed for the efficient production of N-terminal cysteine containing protein fragments for expressed protein ligation (EPL) from polyhistidine-tagged precursors. We provide three examples of efficient CNBr cleavage of fragments of the glycoprotein erythropoietin that can be ligated with peptides or glycopeptide mimetics potentially giving rise to semisynthetic glycoprotein therapeutics.     

β2-微球蛋白连续表位的免疫亲和质谱研究

采用Endoproteinase Glu-C, Lys-C和Trypsin 3种蛋白酶分别水解β2-微球蛋白, 产生一系列肽段, 利用固定在琼脂糖珠上的单克隆抗体与其发生免疫亲和反应. 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术, 对抗原决定簇肽段-抗体复合物进行系统研究, 结果表明, 与抗体结合部位即连续表位的位点为肽段(59~69)(DWSFYLLYYTE). 该研究方法简便、准确, 可用来对其它抗原连续表位的快速测定.     

New approach for measurement of non-SHBG-bound testosterone in human plasma

Testosterone (T) circulates in the blood tightly bound to sex hormone-binding globulin (SHBG) and weakly to albumin. Measuring protein unbound T (free) or non-SHBG-bound T rather than total T has been recommended for the evaluation of androgen disorders in humans. Ammonium sulfate precipitation has been widely used to separate [SHBG-T] complex from free and albumin-bound T. To achieve more specificity in this separation, we used monoclonal anti-SHBG antibody and developed a suitable and convenient immunoassay for measuring non-SHBG-bound T. Magnetic beads were covalently coupled to a monoclonal anti-SHBG antibody to capture [SHBG-T] complex from plasma samples. Magnetic separation was then performed to allow measurement of non-SHBG-bound T in the supernatant by direct radioimmunoassay. When 300 μL of plasma samples were incubated at room temperature with 10 μL of anti-SHBG beads, residual SHBG concentration was undetectable in the supernatant. The specificity of proteins retained on anti-SHBG beads was further demonstrated by peptide mass fingerprint on a MALDI-TOF analyzer. The non-specific adsorption of T on beads was low (5%), and dissociation of T from SHBG-T complex was less than 5% after 180 min of incubation. The plasma concentrations of non-SHBG-bound T using anti-SHBG beads were highly correlated to those obtained using ammonium sulfate precipitation. We conclude that SHBG immunocapture is a highly specific and useful tool for an experimental direct measurement of plasma non-SHBG-bound T. This methodology is also convenient and appropriate for routine and automated assay.     

高灵敏度化学发光磁酶免疫法检测人绒毛膜促性腺激素

将磁性微粒与抗体的偶联,通过优化偶联条件提高了偶联效率,制备了高灵敏度的磁微粒生物探针;采用3-(2-螺旋金刚烷)-4-甲氧基-4-(3-邻氧酰苯基)-1,2-二氧杂环丁烷(AMPPD)--碱性磷酸酶(ALP)化学发光体系,建立了化学发光磁酶免疫检测方法;对检测方法进行了优化和改进,提高了系统的灵敏度和检测速度,并对HCG样品进行相关检测.结果表明,HCG浓度在0.15～150 IU/L范围内,光强随HCG浓度增大而增加,两者之间线性关系良好,相关系数r为0.960;检出限可达0.15 IU/L; 相对标准偏差(RSD)小于5%; 检测总时间少于1 h.本方法可以应用于其它免疫分子的检测,在临床免疫检测方面具有广阔的应用前景.     

Affinity purification of antibodies using immobilized FB domain of protein A.

A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.     

Alkaline phosphatase-labeled macromolecular probe for sensitive chemiluminescence detection of proteins on a solid-phase membrane

In the present study, we synthesized dextran (MW = ca. 2,000 kDa)-based macromolecular probes containing multiple molecules of alkaline phosphatase (ALP) as a signal-trigger enzyme and of biotin as an assembly mediator. The ALP and biotin molecules were covalently attached into the dextran backbone after the formation of aldehyde groups into the macromolecule by periodate oxidation. The synthesized probes contained 27–31 molecules of ALP in their macromolecules when 50-fold molar ratio of ALP to the dextran was used for the synthesis. These probes provided 14–20 times stronger chemiluminescence (CL) than that of the equimolar free ALP adsorbed on a nylon membrane. The velocity of the CL reaction of ALP-catalyzed adamantlyl-1,2-dioxetane substrate was improved from a slower emission (glow type) of CL to a faster one (flash type). The CL signal integrated for 2 min under strongly alkaline conditions (pH 13.0) was about ten times greater than that obtained by the conventional conditions (pH 9.5). Therefore, the synthesized macromolecular probe could be successfully utilized for the high-throughput CL detection of biotin-conjugated anti-rabbit IgG antibody with a lower detection limit of 880 amol per spot on the nylon membrane. This study provides analytical strategy for the rapid, convenient, and sensitive detection of target proteins in immunoassays.     

A Method for the Rapid Discovery of Naturally Occurring Products by Proteins Immobilized on Magnetic Beads and Reverse Affinity Chromatography

A highly efficient screening method for naturally occurring products that bind to a specific target protein was demonstrated by using hVDR magnetic beads. The native ligand 1α,25(OH)₂ VD₃ ( 1 ) was selectively bound by hVDR magnetic beads when present in a mixture of natural compounds. Furthermore, this method was shown to be applicable to the identification of natural products that interact with a specific protein immobilized on the beads from an extract of a natural resource. Two new natural compounds were isolated by this method. This approach will be helpful for the discovery of novel, naturally occurring products that bind to specific target proteins. This method has the further advantages that it can identify the HPLC peak corresponding to the target compound for isolation, as well as provide important UV, CD, or MS profile information.     

"Affinity-proteomics": direct protein identification from biological material using mass spectrometric epitope mapping

We describe here a new approach for the identification of affinity-bound proteins by proteolytic generation and mass spectrometric analysis of their antibody bound epitope peptides (epitope excision). The cardiac muscle protein troponin T was chosen as a protein antigen because of its diagnostic importance in myocardial infarct, and its previously characterised epitope structure. Two monoclonal antibodies (IgG1-1B10 and IgG1-11.7) raised against intact human troponin T were found to be completely cross reactive with bovine heart troponin T. A combination of immuno-affinity isolation, partial proteolytic degradation (epitope excision), mass spectrometric peptide mapping, and database analysis was used for the direct identification of Tn T from bovine heart cell lysate. Selective binding of the protein was achieved by addition of bovine heart cell lysate to the Sepharose-immobilised monoclonal antibodies, followed by removal of supernatant material containing unbound protein. While still bound to the affinity matrix the protein was partially degraded thereby generating a set of affinity-bound, overlapping peptide fragments comprising the epitope. Following dissociation from the antibody the epitope peptides were analysed by matrix assisted laser desorption-ionisation (MALDI) and electrospray-ionisation (ESI) mass spectrometry. The peptide masses identified by mass spectrometry were used to perform an automated database search, combined with a search for a common "epitope motif". This procedure resulted in the unequivocal identification of the protein from biological material with only a minimum number of peptide masses, and requiring only limited mass-determination accuracy. The dramatic increase of selectivity for identification of the protein by combining the antigen-antibody specificity with the redundancy of peptide sequences renders this "affinity-proteomics" approach a powerful tool for mass spectrometric identification of proteins from biological material.     

Single‐Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single‐molecule‐analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

Single-Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single-molecule-analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

On-column capture of a specific protein in capillary electrophoresis using magnetic beads

A method for capturing specific molecules separated by CE has been explored. To demonstrate on-column capture of migrating analyte molecules, two detection windows were fabricated on a capillary. Magnetic beads containing immobilized molecules that react with the specific molecules under study were placed between the detection windows in the capillary using magnets. Molecules in a sample solution injected into the capillary were separated and detected at the first detection window. After passing through the first detection window, the separated molecules encountered the magnetic beads, where the specific analyte was captured. As a result, the peak area for those analyte molecules decreased or disappeared completely at the second detection window. Rabbit IgG and carbonic anhydrase were employed to demonstrate on-column capture of a specific molecule. For rabbit IgG, magnetic beads containing the immobilized antibody (anti-rabbit IgG) were used. Rabbit IgG molecules were captured on the magnetic beads during CE migration. Furthermore, the capture of carbonic anhydrase was demonstrated by the reaction between magnetic beads (containing immobilized anti-rabbit IgG) and anti-carbonic anhydrase (rabbit IgG), before the beads were packed in the capillary. After packing the magnetic beads in the capillary, a mixture of two proteins was injected into the capillary. Two proteins were detected at the first detection window, while the peak corresponding to carbonic anhydrase disappeared at the second detection window. The results show that using an appropriate antibody, the present technique would be applicable to any proteins.     

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.     

Improved in-gel approaches to generate peptide maps of integral membrane proteins with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This paper reports studies of in-gel digestion procedures to generate MALDI-MS peptide maps of integral membrane proteins. The methods were developed for the membrane domain of the mannitol permease of E. coli. In-gel digestion of this domain with trypsin, followed by extraction of the peptides from the gel, yields only 44% sequence coverage. Since lysines and arginines are seldomly found in the membrane-spanning regions, complete tryptic cleavage will generate large hydrophobic fragments, many of which are poorly soluble and most likely contribute to the low sequence coverage. Addition of the detergent octyl-beta-glucopyranoside (OBG), at 0.1% concentration, to the extraction solvent increases the total number of peptides detected to at least 85% of the total protein sequence. OBG facilitates the recovery of hydrophobic peptides when they are SpeedVac dried during the extraction procedure. Many of the newly recovered peptides are partial cleavage products. This seems to be advantageous since it generates hydrophobic fragments with a hydrophilic solubilizing part. In-gel CNBr cleavage resulted in 5-10-fold more intense spectra, 83% sequence coverage, fully cleaved fragments and no effect of OBG. In contrast to tryptic cleavage sites, the CNBr cleavage sites are found in transmembrane segments; cleavage at these sites generates smaller hydrophobic fragments, which are more soluble and do not need OBG. With the results of both cleavages, a complete sequence coverage of the membrane domain of the mannitol permease of E. coli is obtained without the necessity of using HPLC separation. The protocols were applied to two other integral membrane proteins, which confirmed the general applicability of CNBr cleavage and the observed effects of OBG in peptide recovery after tryptic digestion.     

Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 μL min⁻¹ was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.     

C-Terminal sequencing of trichosanthin

Preliminary identification of C-terminus of trichosanthin by chemical and enzymic methods, such as hydrazinolysis, thiohydantoin reaction and carboxypeptidase hydrolysis, showed that there may be the possible presence of more than one terminus, i.e., Met and Ala but complicated by side reactions. A computer-assisted carboxypeptidase method was first introduced by the authors to determine the C-terminal sequence of trichosanthin, and showed that trichosanthin is heterogeneous at its C-terminus and has two C-terminal sequences determined as -Arg-Asn-Asn-Met-OH and -Arg-Asn-Asn-Met-Ala-OH respectively. These results have been later unambiguously confirmed by the results from other experiments through the identification of the free alanine always present in the CNBr degradation products of trichosanthin, and the actual separation of two fragments from the finger prints as well as from the HPLC fractions of the trypsin digest of this protein. All shows that their amino acid sequences, determined by manual DABITC/PITC technique, agree well with those of the two C-terminal sequences determined by the computer-carboxy peptidase method.     

Efficient protein digestion with peptide separation in a micro-device interfaced to electrospray mass spectrometry

A highly efficient protein digestion device has been fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected either directly to an electrospray mass spectrometer via a 'microtight T' connector, from which aqueous acetic acid (0.2%) was pumped, or via a monolithic column connected to the mass spectrometer ion source. Six proteins with molecular mass ranging from 2848 to 77703 Da were digested completely using this system. In the second set of experiments a short monolithic separation column was placed after the immobilized trypsin capillary and partial separation of the generated peptides was obtained. The detection limits were increased from the micromol to pmol range by utilization of this separation column. Gradient elution, using a binary HPLC pump and a flow splitter, was used to optimize the peptide separation. This provided significantly enhanced resolution of the tryptic peptides but increased the analysis time to 30 minutes.