Application of phenylboronic acid modified hydrogel affinity chips for high-throughput mass spectrometric analysis of glycated proteins

The complexity of the human plasma proteome is greatly increased by post-translational modifications. Besides physiological modifications, pathological conditions such as diabetes are responsible for adding to this complexity by producing advanced glycation endproducts (AGEs). When searching for specific biomarkers it is a prerequisite to reduce this complexity prior to analysis. To do this, agarose hydrogel was used to create a high-capacity affinity layer on the modified aluminum surface of MALDI (matrix-assisted laser desorption/ionization) targets. 3-Aminophenylboronic acid was immobilized via cyanogen bromide activation as a ligand for affinity sorption of glycated proteins, followed by their direct detection by MALDI. High protein capacity of prepared MALDI chips, efficient separation and low non-specific protein binding were demonstrated. The results show that phenylboronic acid modified hydrogels are very suitable for creating affinity surfaces for the high-throughput analysis of AGEs.     

Immobilization and surface characterization of NeutrAvidin biotin-binding protein on different hydrogel interlayers

For a number of potential applications, it is desirable to immobilize avidin class molecules onto solid supports and exploit their ability to bind biotinylated molecules with high affinity. NeutrAvidin molecules were surface immobilized in various ways. In this study, NeutrAvidin was covalently attached by carbodiimide chemistry onto carboxyl groups of polyacrylic acid and carboxymethyl-dextran hydrogel interlayers. A third strategy involved the affinity "docking" of NeutrAvidin onto a biotinylated poly(ethylene glycol) interlayer. These three interlayers were selected for their low nonspecific binding of proteins, which was expected to minimize surface binding of NeutrAvidin by nonspecific interfacial adsorption. X-ray photoelectron spectroscopy (XPS) analyses allowed detailed characterization of the multilayer fabrication steps. An ELISA assay was used to measure NeutrAvidin activity, which varied with the surface immobilization route. Atomic force microcopy (AFM) force measurements showed that the hydrogel interlayer contributed to a repulsive force and verified the specific interaction between biotinylated AFM tips and the NeutrAvidin surfaces. When a solution of free biotin was injected into the AFM liquid cell, the force curve changed substantially and became identical to that recorded between surfaces carrying no NeutrAvidin, indicating that the free solution biotin had displaced NeutrAvidin proteins off the PEG-biotin layer.     

Hollow-fiber flow field-flow fractionation: a gentle separation method for mass spectrometry of native proteins

Low-impact ionization sources like electrospray ionization (ESI) and matrix-assisted, laser desorption/ionization (MALDI) equipped with time-of-flight (TOF) mass analyzers provide intact protein analysis over a very wide molar mass range. ESI/TOFMS provides also indications on the higher-order structure of intact proteins and non-covalent protein complexes. However, direct analysis of intact proteins mixtures in real samples shows limited success, mainly because spectra become very complex to interpret. This is also due to sample contaminants, and to the mechanism of competitive ionization in ESI or MALDI. Rapid and efficient sample clean-up and separation methods can significantly enhance the power of TOFMS for intact protein analysis. However, if protein native conditions want to be maintained, the methods should affect neither the three-dimensional structure nor the non-covalent chemistry of the proteins. Reversed-phase (RP) HPLC, size-exclusion chromatography (SEC), and capillary zone electrophoresis (CZE) are on-line or off-line coupled to ESI/TOFMS or MALDI/TOFMS. In fact, these separation methods often show limitations when applied to the analysis of native proteins. Organic modifiers and saline buffers are required in the case of RP HPLC or CZE. They can induce protein degradation or affect ionization when MS is performed after separation. High voltages used in CZE can contribute to alter proteins from their native form. In the case of high molar mass proteins, SEC is scarcely selective, and barely able to detect protein aggregates. Sample entanglement/adsorption on the stationary phase can also occur.     

Cysteine‐capped ZnSe quantum dots as affinity and accelerating probes for microwave enzymatic digestion of proteins via direct matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis

Fluorescent semiconductor quantum dots (QDs) exhibit great potential and capability for many biological and biochemical applications. We report a simple strategy for the synthesis of aqueous stable ZnSe QDs by using cysteine as the capping agent (ZnSe‐Cys QDs). The ZnSe QDs can act as affinity probes to enrich peptides and proteins via direct matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) analysis. This nanoprobe could significantly enhance protein signals (insulin, ubiquitin, cytochrome c, myoglobin and lysozyme) in MALDI‐TOFMS by 2.5–12 times compared with the traditional method. Additionally, the ZnSe‐Cys QDs can be applied as heat absorbers (as accelerating probes) to speed up microwave‐assisted enzymatic digestion reactions and also as affinity probes to enrich lysozyme‐digested products in MALDI‐TOFMS. Furthermore, after the enrichment experiments, the solutions of ZnSe‐Cys QDs mixed with proteins can be directly deposited onto the MALDI plates for rapid analysis. This approach shows a simple, rapid, efficient and straightforward method for direct analysis of proteins or peptides by MALDI‐TOFMS without the requirement for further time‐consuming separation processes, tedious washing steps or laborious purification procedures. The present study has demonstrated that ZnSe‐Cys QDs are reliable and potential materials for rapid, selective separation and enrichment of proteins as well as accelerating probes for microwave‐digested reactions for proteins than the regular MALDI‐MS tools. Additionally, we also believe that this work may also inspire investigations for applications of QDs in the field of MALDI‐MS for proteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

improvement of the Specificity of Surface Plasmon Resonance with BSA-modified Chip

Recently, surface plasmon resonance (SPR) become more and more popular without the need of the label technology1-3. However, sometimes, a number of experimental artifacts complicate the final biosensor analysis4-7. The utilization of a reference surface c…     

Accurate mass measurement of DNA oligonucleotide ions using high-resolution time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) time-of-flight mass spectrometry (TOFMS) play an essential role in the analysis of biological molecules, not only peptides and proteins, but also DNA and RNA. Tandem mass spectrometry used for sequence analysis has been a major focus of technological developments in mass spectrometry, but accurate mass measurements by high-resolution TOFMS are equally important. This paper describes the role that high mass measurement accuracy can play in DNA composition assignment and discusses the influence of several parameters on mass measurement accuracy in both MALDI and ESI mass spectra. Five oligonucleotides (5-13mers) were used to test the resolving power and mass measurement accuracy obtained with MALDI and ESI instruments with reflectron TOF mass analyzers. The results from the experimental studies and additional theoretical calculations provide a basis to predict the practical utility of high-resolution TOFMS for the analysis of larger oligonucleotides.     

Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.     

Phenylboronic acid self-assembled layer on glassy carbon electrode for recognition of glycoprotein peroxidase

We have successfully fabricated a phenylboronic acid self-assembled layer on glassy carbon electrodes (GCE), where 3-aminophenylboronic acid (APBA) is covalently bound to the electrochemical pretreated GCE surface with glutaraldehyde linkage. The specific binding of glycoprotein peroxidase with the self-assembled layer has been studied using horseradish peroxidase (HRP) as a model glycoprotein. Cyclic voltammetric, electrochemical impedance studies and photometric activity assays show that the affinity interaction of HRP with the APBA modified GCE surface includes specific and nonspecific bonding. The specific binding is attributed to the boronic acid–diols interaction where the boronic acid specifically binds the glycosylation sites of the HRP. This specific binding is reversible and can be split by sorbitol and glucose or released in an acidic buffer. The catalytic current of the HRP-loaded electrode, due to the catalytic oxidation of thionine in the presence of hydrogen peroxide, is proportional to HRP concentrations of the incubation solution. This work offers a new way to build novel sensors by specific binding of glycoproteins to a boronic acid self-assembled layer for determination of glycated proteins.     

Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial beta-elimination/chemical tagging and matrix-assisted laser desorption/ionization mass spectrometric analysis

The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data.     

单克隆抗体1F2的制备、纯化及质谱测定

用小分子meso -四 (α,α ,α ,α -O -苯乙酰苯 )卟啉免疫Balb/c小鼠 ,用单克隆抗体技术得到细胞株 1F2和单抗 1F2 ,这是对传统免疫理论的一个突破 .利用高效液相色谱和基质辅助激光解吸质谱 (matrixassistedlaserdesorptionionizationtime_of_flightmassspectography ;缩写为MALDI/TOFMS)证明了纯化得到单抗体 1F2的纯度很好 ,同时得到的单抗 1F2的相对分子量为 15 6 6 78.8Da .MALDI/TOFMS提供了一种测定蛋白质相对分子量和纯度快速准确的方法     

Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: role of water activity

Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for immobilized metal-chelate affinity adsorbents. Addition of 20% (v/v) of solutes such as ethanol, methanol, isopropanol, n-propanol, and dimethyl sulfoxide enhances the initial affinity of binding of total yeast RNA by 4.4-, 3.8-, 3.7-, 3.0-, and 2.8-fold, respectively for Cu(II)-iminodiacetic acid (IDA) agarose adsorbent, and the weaker adsorption by Cu(II)-nitrilotriacetic acid (NTA) agarose was even more strongly enhanced. The adsorption affinities of the smaller oligodeoxynucleotide molecules A20, G20, C20 and T20 also increase with the addition of ethanol, suggesting that the effect is not significantly mediated by conformational changes. Binding enhancement generally correlates with reduction of water activity by the various solutes, as predicted by several models of solution thermodynamics, consistent with an entropic contribution by displacement of waters from the metal-chelate. Interestingly, the enhancement was not seen with the proteins bovine serum albumin and lysozyme.     

Micropatterning of proteins on the surface of three-dimensional poly(ethylene glycol) hydrogel microstructures

This paper describes micropatterning of proteins on the surface of three-dimensional hydrogel microstructures. Poly(ethylene glycol) (PEG)-based hydrogel microstructures were fabricated on a glass substrate using a poly(dimethylsiloxane) (PDMS) replica as a molding insert and photolithography. The lateral dimension and height of the hydrogel microstructures were easily controlled by the feature size of the photomask and depth of the PDMS replica, respectively. Bovine serum albumin (BSA), a model protein, was covalently immobilized to the surface of the hydrogel microstructure via a 5-azidonitrobenzoyloxy N-hydroxysuccinimide bifunctional linker at a surface density of 1.48 mg cm⁻². The immobilization of BSA on the PEG hydrogel surface was demonstrated with XPS by confirming the formation of a new nitrogen peak, and the selective immobilization of fluorescent-labeled BSA on the outer region of the three-dimensional hydrogel micropattern was demonstrated by fluorescence. A hydrogel microstructure could immobilize two different enzymes separately, and sequential bienzymatic reaction was demonstrated by reacting glucose and Amplex Red with a hydrogel microstructure where glucose oxidase was immobilized on the surface and peroxidase was encapsulated. Activity of immobilized glucose oxidase was 16.5 U mg⁻¹, and different glucose concentration ranged from 0.1 to 20 mM could be successfully detected.     

Glass-chip-based sample preparation and on-chip trypic digestion for matrix-assisted laser desorption/ionization mass spectrometric analysis using a sol-gel/2,5-dihydroxybenzoic acid hybrid matrix

A glass-chip-based sample preparation method for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis of tryptic digests of proteins and intact cells is described. A MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), was hybridized with sol-gels to generate a sol-gel-derived material. Taking advantage of the characteristics of sol-gels, the sol-gel-derived material readily adhered to the surface of a glass chip through covalent bonding. Only one step of sample preparation, deposition of the sample solution on the glass chip, was required before MALDI-MS analysis. Because 2,5-DHB was homogeneously dispersed on the sol-gel network structure, good spot-to-spot reproducibility was obtained in MALDI analysis using this approach and the analyte signals were uniform throughout the chip. The modified glass chips were robust and effective for at least 1 week. This glass-chip-based matrix preparation method provides a straightforward approach to developing techniques for analyzing the on-chip enzymatic digestion of proteins and intact cells of microorganisms. Cytochrome C and Escherichia coli were used as analytes to demonstrate the feasibility of this approach. The products of the on-chip enzymatic digests were identified through protein database searches.     

Immobilization of RNase S-Peptide on a single-stranded DNA-fixed gold surface and effective masking of its surface by an acridinyl poly(ethylene glycol)

Oligonucleotide-peptide conjugate was synthesized by coupling of RNase S-peptide to a 24-mer single-stranded DNA (ssDNA) oligonucleotide to be immobilized on its complementary ssDNA oligonucleotide-fixed gold surface of sensor chip or electrode. Immobilization of on the ssDNA-fixed gold surface through DNA duplex formation was confirmed by quartz crystal microbalance (QCM) and electrochemical measurements. After treating with a synthetic acridinyl poly(ethylene glycol) (APEG), specific interaction of S-protein with the S-peptide immobilized on the gold surface was demonstrated by QCM without nonspecific adsorption of unrelated proteins such as BSA and RNase A at the surfaces. This result suggested that the acridine parts of APEG could bind to the DNA duplex on the gold surface and the poly(ethylene glycol) parts were fastened on the surface to resist the adsorption of proteins. Thus, the combination of oligonucleotide-peptide conjugate, ssDNA-fixed chip and APEG with effective masking property provides a new tool for the analysis of specific peptide-protein interactions without disturbance by other unrelated proteins.     

High affinity capture surface for matrix-assisted laser desorption/ionisation compatible protein microarrays

A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionisation (MALDI) targets has been investigated. Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry. Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule. Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E. coli. The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target. Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E. coli proteins were detected. Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins. Furthermore, protein-protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated. Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array. The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins. Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry.     

固定化肌红蛋白在水-乙醇混合溶液中的类酶活性电化学

用琼脂糖将肌红蛋白(Mb)固定到玻碳电极(GC)表面,制备了Mb-琼脂糖膜修饰电极。包埋在琼脂糖膜中的Mb在缓冲溶液和乙醇混合溶液中与电极直接传递电子,得到一对对称的Mb辅基血红素Fe(III)/Fe(II)电对的可逆氧化还原峰。其式电势随缓冲溶液pH值增加而负移,且呈线性关系,这说明Mb的电子传递过程伴随有质子的转移。在缓冲溶液和乙醇混合溶液中,固定化肌红蛋白表现出类似细胞色素P450的催化活性,能快速催化还原氯乙烷(六氯乙烷、五氯乙烷、四氯乙烷)脱氯,Mb-琼脂糖膜修饰电极具有较好的稳定性和重现性,可用于这些物质的定量检测。     

Immobilization of aminophenylboronic acid on magnetic beads for the direct determination of glycoproteins by matrix assisted laser desorption ionization mass spectrometry

Aminophenylboronic acid (APBA) has been immobilized on magnetic beads for the direct determination of glycoprotein by matrix assisted laser desorption/ionizaton time of flight mass spectrometry (MALDI-TOF-MS). An APBA layer was formed on the surface of carboxylic acid terminated magnetic beads by coupling with carbodiimide and subsequently reacted with an N-hydroxysuccinimide moiety. The immobilized APBA was identified by MALDI-TOF-MS without a matrix. Glycoproteins, such as HbA1c, fibrinogen, or RNase B were separated and desalted using APBA magnetic beads by simply washing the magnetic beads and then separating them by external magnet. Proteins can be identified by direct determination of proteins on beads on MALDI plate and confirmed again by peptide mass finger printing after digestion of proteins on magnetic beads by trypsin. Fluorescence image with a FITC tagging protein using confocal laser microscopy showed the difference of immobilization efficiency between glycoproteins and nonglycoproteins. The methods developed within this work allow the simple treatment and enrichment of glycoproteins as well as direct determination of proteins on beads by MALDI-TOF-MS.     

Comprehensive analysis of glycated human serum albumin tryptic peptides by off-line liquid chromatography followed by MALDI analysis on a time-of-flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.     

Photocleavable peptide hydrogel arrays for MALDI-TOF analysis of kinase activity

We have developed an acrylamide copolymerization strategy to immobilize acrylamide labeled peptides and proteins into a hydrogel surface and detect their modifications using MALDI-TOF mass spectrometry. Copolymerization into hydrogels is robust, compatible with "off-the-shelf" chemistry, and yields materials and surfaces that are stable to aqueous or organic solvents, drying, high or low temperature, high or low pH, oxidizing agents, sonication, mechanical contact, etc. The use of acrylamide hydrogels allows immobilization of substrates in a hydrated environment that can be used both as a biological reaction matrix and as a MALDI target. In our strategy, a substrate peptide was designed in a modular fashion to include both modification site and affinity domains. It was labeled with an acrylamide functionality using a generalized chemistry and covalently attached to the surface with a photocleavable linker, allowing for aggressive washing to remove any fouling, followed by selective release for MALDI-TOF analysis. Using this system we were able to analyze and compare v-Abl (truncated) and c-Abl (full-length) kinase activity on a peptide substrate with an affinity domain specific for the full-length kinase, observing excellent overall reproducibility in the extent of phosphorylation detected. This work serves as proof of principle for modular substrate design strategies for mass spectrometry-readable biosensors.     

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.