The Bead blot: a method for selecting small molecule ligands for protein capture and purification

We present a new method for selecting peptide ligands that are useful for protein purification, protein targeting and exploring protein-ligand interactions, and which requires no prior protein purification or derivatization. In the Bead blot, a complex mixture containing the target protein, for example, plasma, is incubated with a combinatorial library of peptide ligands synthesized on beads. The proteins are fractionated and purified on their respective ligands and the beads with their bound proteins are immobilized in a gel. The proteins are eluted from the ligands by capillary action and captured on a membrane so that their position on the membrane corresponds to the position of the beads in the gel. The protein is detected on the membrane, generating spots on an autoradiography film, the spots on the film are aligned with the beads in the gel, and the beads that bound the protein are recovered. The ligand on the bead(s) can be sequenced and synthesized at large scale for protein purification. The Bead blot can be completed in several hours with overnight pause steps if desired. On average, 5 prospective ligands are selected per 50,000 beads screened using this method. Unlike other ligand identification methods, the target protein does not have to be purified or labeled, and the Bead blot allows ligands for multiple proteins to be selected in a single experiment.     

Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.     

A method for probing the affinity of peptides for amphiphilic surfaces

We have developed a rapid method for probing the affinity of peptides toward an amphiphilic surface. Hydrophobic polystyrene-divinylbenzene beads of 5.7 +/- 1.5 micron diameter are coated with a monomolecular film of egg lecithin to achieve the equilibrium spreading density of the phospholipid, 6 X 10(-3) molecule/A2. The coated beads are ideally suited for assessing the affinity of peptides for phospholipid surfaces: Large quantities of lipid-coated beads of known surface area can be prepared easily and rapidly. Within the pH range 2.0 to 9.0, the adsorbed phospholipids are relatively resistant to hydrolysis and remain bound indefinitely. Following incubation with peptide ligands, beads can be separated from the reaction mixture by centrifugation. Peptides, such as melittin, which destroy or cause fusion of single bilayer phospholipid vesicles, cannot disrupt lecithin-coated beads in a comparable way, and do not displace lecithin from the surface of beads. After incubating these beads in solutions of peptides and proteins, we have determined the parameters for the binding of several ligands to the phospholipid surface. The binding of many amphiphilic peptides obeys a Langmuir adsorption isotherm, i.e., saturable reversible binding to independent and equivalent sites on the bead. That the binding is a true reversible equilibrium is shown by desorption of the ligand upon dilution. From the isotherm, the surface areas occupied by the ligand molecules were calculated, and were observed to be similar to those observed in monolayers at the air-water interface. In comparing the binding of amphiphilic peptides to that of completely hydrophilic peptides, we observed that only the former bind at levels measurable by our techniques. Thus, this method can serve as a rapid assay for detecting amphiphilicity in peptides of putative amphiphilic character.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.     

Solid-Phase Nested Deletion: A New Subcloning-less Method for Generating Nested Deletions

We have developed a new subcloning-less method for generatingnested deletions which we have termed Solid-Phase Nested Deletion.The basic procedure for this method is as follows. The targetDNA fragment is cloned in the multiple cloning site of a cloningvector, pUC or its derivatives, and amplified by PCR using aset of primers, one of which is 5'-biotinylated. The amplifiedDNA is partially digested by a restriction enzyme with a 4-baserecognition sequence. The digested DNA is ligated with a syntheticadapter DNA. Monodiverse beads coupled with streptavidin (Dynabeads^TMM-280 streptavidin) are added to the mixture and the biotinylatedDNA fragments are separated by applying magnetic field. Theunidirectionally deleted DNA fragments are recovered by PCRfrom the magnetic beads, and size-fractionated by agarose gelelectrophoresis. The DNA fragments are amplified by PCR andused for sequencing. We demonstrate the potential of this methodusinga 4878-bp EcoRI fragment of phage DNA.     

Enzymatic replacement of RNA sequences in vitro; use of FPLC for rapid and efficient preparation of tRNA variants

A chromatographic procedure using columns of Superose-12 (gel filtration) and PBA-30 (phenylboronate agarose) in tandem has been developed. This procedure is very useful for rapidly separating and efficiently recovering each of the components in the reaction mixture of joining reaction with RNA ligase, and thereby is also beneficial to rapid preparation of tRNA variants in a high yield.     

Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides

Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.     

Selective enrichment of tryptophan-containing peptides from protein digests employing a reversible derivatization with malondialdehyde and solid-phase capture on hydrazide beads

A method for the selective enrichment of tryptophan-containing peptides from complex peptide mixtures such as protein digests is presented. It is based on the reversible reaction of tryptophan with malondialdehyde and trapping of the derivatized Trp-peptides on hydrazide beads via the free aldehyde group of the modified peptides. The peptides are subsequently recovered in their native form by specific cleavage reactions for further (mass spectrometric) analysis. The method was optimized and evaluated using a tryptic digest of a mixture of 10 model proteins, demonstrating a significant reduction in sample complexity while still allowing the identification of all proteins. The applicability of the tryptophan-specific enrichment procedure to complex biological samples is demonstrated for a total yeast cell lysate. Analysis of the processed fraction by 1D-LC-MS/MS confirms the specificity of the enrichment procedure, as more than 85% of the peptides recovered from the enrichment step contained tryptophan. The reduction in sample complexity also resulted in the identification of additional proteins in comparison to the untreated lysate.     

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.     

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity

ABSTRACT

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method’s high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.     

Selective enrichment of specific DNA, cDNA and RNA sequences using biotinylated probes, avidin and copper-chelate agarose.

We have developed a general procedure for the rapid and efficient enrichment of specific DNA, RNA or cDNA sequences. Biotinylated DNA or RNA is used as a hybridization probe in solution, avidin is then added to label both the probe and hybrid molecules, and the hybridization mixture chromatographed over cupric-iminodiacetic acid agarose beads. Avidin-probe and avidin-hybrid molecules are selectively retained on the column; non-hybridized sequences are contained in the flow-through fraction. Sequences retained on the column are recovered in high yield by the addition of ethylenediamine tetracetic acid in the buffer. The method can be used in both subtractive enrichment and positive selection protocols. Here we report its application to the isolation of Neisseria gonorrhoeae specific genomic DNA clones and the purification of a cDNA subpopulation representing mRNA sequences that are over-expressed in murine Friend cells after dimethylsulfoxide induction.     

An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture

A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.     

Use of magnetic carboxyl beads to purify a cationic peptide in a batch system

Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic peptides are purified by size exclusion chromatography or ion exchange chromatography, neither of which is easily applicable to small volumes of diluted peptide samples. We developed a small-scale system that is easily adaptable for high-throughput screening and uses carboxyl magnetic beads to purify a cationic peptide from its fusion partner.     

Estimation of cell surface associated protease activity and its application to lymphocytes

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene-divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells. This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three- to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37° C have enzymatically altered surface properties. Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid-phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.     

A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.     

High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glass beads

We describe here a reliable high-throughput method for extraction of RNA from fresh or frozen plant tissue that obviates laborious and time-consuming homogenisation by mortar and pestle. The method is based on homogenisation by high-speed reciprocal shaking in presence of a mixture of inexpensive abrasive materials; i.e., quartz sand and glass beads. After homogenisation, the method follows a standard procedure for RNA extraction by phenol/LiCl. Yield and quality of RNA obtained by homogenisation with the sand/glass bead mix are identical to those obtained by mortar and pestle.     

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.     

Solid-phase sequencing on the gas-phase sequencer

Automated Edman degradation has been successfully used for determining the primary structure of numerous peptides and proteins. Quantitative solid-phase Edman degradation has great potential use for amino acid sequence analysis of synthetic peptides assembled on resin support by the Merrifield procedure. We report here the combined use of a modified gas-phase sequencer program and our improved reversed-phase HPLC analysis for PTH-amino acids to carry out the sequence analysis on synthesized peptide resins. This approach is far more sensitive than using glass beads on the conventional solid-phase sequencer. The peptide was assembled on copoly (styrene-1% divinylbenzene) resin beads at an initial substitution of 0.54 mmol/g. On a routine basis, 10-15 resin beads are used, and a repetitive yield of 94% is obtained: as few as 4 beads can be successfully sequenced. The HPLC PTH-amino acid analysis is sensitive down to subpicomole quantities. This procedure offers a sensitive and rapid analytical tool for checking the purity of peptides as they are being assembled on solid support.     

Removal of mixtures of acetaldehyde and propionaldehyde from waste gas in packed column with immobilized activated sludge gel beads

The removal of mixed acetaldehyde and propionaldehyde as a model of the binary contaminants in waste gas was studied in the packed column containing the immobilized activated sludge gel beads together with the hollow plastic balls developed for the removal of a single aldehyde in the previous work. The rate of each aldehyde biodegradation by the gel beads in the aldehydes mixture was expressed by the Michaelis-Menten type rate equation with an inhibitory term due to the other coexistent aldehyde. The kinetic parameters involved were found to be the same as those determined previously for biodegradation of a single aldehyde. A model for prediction of removal of each aldehyde in the packed column was developed assuming that each aldehyde dissolved in the aqueous phase within the gel bead was biodegraded according to the above rate equation with no mass transfer effect. The packed column was stable and efficient for removal of the binary aldehydes mixture with a very low pressure drop for gas flow due to a reduced gel beads bed compaction by the hollow plastic balls. Removal of each aldehyde decreased with increasing the inlet aldehyde concentrations since each biodegradation rate itself approached asymptotically the maximum one with increase in each aldehyde concentration. The observed removals for each aldehyde in the aldehydes mixture agreed well with those calculated from the design equations developed. The contact efficiency of gel beads with the waste gas stream was estimated to be the same value of 0.24 as in the previous work, supporting that the efficiency was specific to the geometrical and physical properties of the packed column used.     

Purification, amino terminal analysis, and peptide mapping of proteins after in situ postelectrophoretic fluorescent labeling

Proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained in situ with either 5-(dimethylamino)-1-naphthalene sulfonyl chloride (dansyl chloride) or fluorescein isothiocyanate. This staining procedure can be carried out in less than 30 min without previous fixation of the proteins. It is not dependent on such factors as charge or molecular weight of the proteins and can detect 50 ng of protein in a 10-mm-wide gel slot. Fluorescent staining with dansyl chloride was used to localize proteins after electrophoresis for subsequent electroelution, amino terminal analysis, and peptide mapping. The electroelution can be carried out in less than 3 h with yields approaching 100%. The staining of only one strip of a preparative gel allowed the electroelution of proteins without covalent modification. For amino terminal analysis, identical results were obtained when the hydrolysis step was carried out after electroelution or directly in the gel pieces. The peptide mapping can be carried out with the proteins in solution (after electroelution) or directly in the gel pieces. The amino terminal and peptide mapping analysis of each protein in a mixture can be completed within 30 h from the beginning of the electrophoretic fractionation. The method appears to be applicable to a wide range of proteins showing very different biochemical properties.