The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Gar&#231;on. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).

1. Preparation of Various Solutions and Protein Samples
<ol>
	<li>Prepare 100 ml of phosphate buffer solution (PBS-G) containing 50 mM NaH2PO4, 50 mM NaCl, and 10% glycerol at pH 6.8.</li>
	<li>Prepare 250 ml of HEPES buffer solution containing 10 mM HEPES, 150 mM NaCl, 0.005% Tween 20 at pH 7.4.</li>
	<li>Prepare stock solution of building block 1 (BB1) lactose and building block 2 (BB2) sulfated lactose (Figure 1)&#160;at 0.2 mM in PBS-G.</li>
	<li>Prepare 1 ml of protein solutions in HEPES: Arachis hypogaea lectin (AHL) at 500 nM, myoglobin at 1 &#181;M, and lysozyme at 500 nM.</li>
	<li>Prepare 20 ml of 1% SDS in ultrapure water.</li>
</ol>
2. Preparation of the CoCRR Array
<ol>
	<li>Clean the gold surface of the prism 48 hr prior to use with a plasma cleaner for 3 min under these conditions: 75% Oxygen, 25% Argon, 0.6 mbar, power 40 W.</li>
	<li>Prepare eleven pure and mixed solutions of BB1 and BB2 with [BB1]/([BB1]+[BB2]) ratios between 0 and 100% by increments of 10% at a total BB concentration of 0.1 mM in PBS-G.</li>
	<li>Deposit 8 nl droplets of these pure and mixed solutions on the prism surface using a non-contact spotter in quadruplicate for each ratio with 44 spots in total (Figure 2). 
	NOTE: 10% glycerol added in PBS-G is very important to reduce solvent evaporation and the change of BB concentration after deposition.</li>
	<li>Place the prism inside a Petri dish containing 1 ml of ultrapure water and leave it O/N at RT for self-assembly of BB1 and BB2 on the gold surface. Herein, it is assumed that the average surface composition in the mixed SAMs reflects the composition of the deposition mixed solution (Figure 3)11.</li>
	<li>Wash thoroughly the prism with ultrapure water and then dry it under a flow of argon.</li>
</ol>
3. Protein Sensing by SPRi
<ol>
	<li>Set the incubator in which the SPRi apparatus is placed at 25 &#176;C to avoid refractive index changes induced by temperature variation during protein sensing.</li>
	<li>Insert a non-functionalized rinsing prism in the 10 &#181;l Polyether ether ketone (PEEK) flow cell connected to a computer controlled syringe pump, a degasser and a 6-port medium pressure injection valve (Figure 4). Fill in the flow system with freshly filtered and degassed running buffer HEPES.</li>
	<li>Remove the rinsing prism and insert the prism containing the CoCRR array in the flow cell. Run HEPES at a 100 &#181;l/min flow rate. With the help of the CCD camera remove any air bubbles present on the prism surface by passing running buffer quickly.</li>
	<li>Define study area for each spot by drawing a circle with the same diameter for the area of interest based on a well-contrasted image of the array and with the help of integrated software in SPRi system.</li>
	<li>Trace plasmon curves, which represent reflectivity curves in function of the incident angle, for all the spots.</li>
	<li>Choose the working angle (position at which kinetic curves will be recorded) at the highest slope of the reflectivity curves. Rotate the scanning mirror to fix the selected working angle for kinetic measurements.</li>
	<li>Continue to run HEPES through the flow system until reflectivity signal for all the spots is stable and constant.</li>
	<li>Inject 1 ml AHL solution with a syringe into the sample loop (500 &#181;l) with the injection valve on position &#8220;load&#8221;. Then put the injection valve to position &#8220;injection&#8221;. The flow rate used for all the protein injection was 100 &#181;l/min.</li>
	<li>Start kinetic measurements by monitoring reflectivity variations against time simultaneously on all the spots.</li>
	<li>At the end of protein injection, rinse the array with running buffer for 8 min. Finally, inject 1 ml 1% SDS to regenerate it.</li>
	<li>Repeat the same procedure for the other proteins.</li>
</ol>
4. Data Processing and Analysis
<ol>
	<li>Following entry of protein solution into the flow cell, molecular binding occurs and induces a shift of the plasmon curves and a variation of reflectivity. The image acquisition software converts the measured light intensity values to gray scale levels, giving SPR images Figure 5A, and generates the variation of reflectivity versus time, giving sensorgrams (Figure 5B).</li>
	<li>Use a math computing software to plot the reflectivity (R%) at the end of each protein injection versus the [BB1]/([BB1]+[BB2]) ratio to generate 2D continuous evolution profile (CEP) for each sample (Figure 5C).</li>
	<li>Add the [BB1]/([BB1]+[BB2]) ratio in sensorgrams (Figure 5B)&#160;to generate 3D continuous evolution landscape (CEL) for each protein (Figure 5D).</li>
</ol>

<ol>
	<li>Margulies, D., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+protein+recognition+as+an+alternative+approach+to+antibody-mimetics.">Combinatorial protein recognition as an alternative approach to antibody-mimetics.</a> Current Opinion in Chemical Biology. 14, 705-712 (2010).</li><li>Umali, A. P., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+approach+to+differential+sensing+using+synthetic+molecular+receptors.">A general approach to differential sensing using synthetic molecular receptors.</a> Current Opinion in Chemical Biology. 14, 685-692 (2010).</li><li>Baldini, L., Wilson, A. J., Hong, J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-based+detection+of+different+proteins+using+an+array+of+fluorescent+protein+surface+receptors.">Pattern-based detection of different proteins using an array of fluorescent protein surface receptors.</a> J. Am. Chem. Soc. 126, 5656-5657 (2004).</li><li>Zhou, H. C., Baldini, L., Hong, J., Wilson, A. J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+of+proteins+based+on+an+array+of+functionalized+porphyrins.">Pattern recognition of proteins based on an array of functionalized porphyrins.</a> J. Am. Chem. Soc. 128, 2421-2425 (2006).</li><li>Wright, A. T., Griffin, M. J., Zhong, Z., McCleskey, S. C., Anslyn, E. V., McDevitt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptors+create+patterns+that+distinguish+various+proteins.">Differential receptors create patterns that distinguish various proteins.</a> Angew. Chem. Int. Ed. 44, 6375-6378 (2005).</li><li>Wright, A. T., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptor+arrays+and+assays+for+solution-based+molecular+recognition.">Differential receptor arrays and assays for solution-based molecular recognition.</a> Chem. Soc. Rev. 35, 14-28 (2006).</li><li>You, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+proteins+using+nanoparticle&#8211;fluorescent+polymer+&#8216;chemical+nose&#8217;+sensors.">Detection and identification of proteins using nanoparticle&#8211;fluorescent polymer &#8216;chemical nose&#8217; sensors.</a> Nature Nanotechnology. 2, 318-323 (2007).</li><li>De, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+proteins+in+human+serum+using+conjugates+of+nanoparticles+and+green+fluorescent+protein.">Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein.</a> Nature Chemistry. 1, 461-465 (2009).</li><li>Hou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+evolution+profiles+for+electronic-tongue-based+analysis.">Continuous evolution profiles for electronic-tongue-based analysis.</a> Angew. Chem. Int. Ed. 51, 10394-10398 (2012).</li><li>Campbell, C. T., Kim, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPR+microscopy+and+its+applications+to+high-throughput+analyses+of+biomolecular+binding+events+and+their+kinetics.">SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics.</a> Biomaterials. 28, 2380-2392 (2007).</li><li>Stranick, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer-scale+phase+separation+in+mixed+composition+self-assembled+monolayers.">Nanometer-scale phase separation in mixed composition self-assembled monolayers.</a> Nanotechnology. 7, 438-442 (1996).</li></ol>

The authors have no conflicts of interest to declare.

The most critical steps for construction of this eT are dedicated to ensure good reproducibility of the system. For example, cleaning the gold surface of the prism with a standardized procedure before use, adding 10% of glycerol in the eleven pure and mixed solutions of BB1 and BB2 to eliminate solvent evaporation during BBs self-assembling on the gold surface of the prism, depositing multiple replicates for each [BB1]/([BB1]+[BB2]) ratio,&#160;etc. As for protein sensing by SPRi, the choice of the working angle...

Precise and rapid protein sensing methods are very important in medical diagnostics and proteomics. Classical protein-detecting arrays, such as biochips, are based on the &#8220;lock-and-key&#8221; recognition principle and require specific receptors such as aptamers, antibodies, or mimetics.
In recent years, differential sensing inspired by the human olfaction and gustation has emerged as an alternative1. This electronic nose/tongue (eN/eT) approach is based on differential binding of analytes to an array of cross-reactive receptors (CRRs), that do not need to be highly specific or selective for the target molecules thus allow to surmount the laborious process of developing highly selective receptors. It is the combined response of all the receptors that creates a distinct pattern for each sample, like a fingerprint, allowing its identification.
The two key challenges for the development of electronic nose/tongue for effective protein sensing are the production of sensing elements that have the ability to distinguish among structurally similar analytes and the appropriate transduction system for the binding event. Up to now, studies have reported various approaches to array development2. For example in one study an array-based identification of proteins was developed using CRRs prepared from tetra-carboxyphenylporphyrin derivatives by coupling the carboxyl groups to various amino acids or dipeptides to provide differential receptors possessing a hydrophobic core for affinity for proteins and distinct charged peripheries for imparting differential binding. Using this system, different proteins and protein mixtures were identified by measuring fluorescence quenching of the receptors upon interaction with the analytes3,4. In another study, a library of 29 CRRs containing tripeptide and boronic acid moieties synthesized in a combinatorial way on a hexasubstituted benzene scaffold was developed for sensing proteins with an indicator-uptake colorimetric detection5,6. With such a design, each receptor showed differential binding capacity with proteins based on the variance in the peptide arms, and the boronic acids assisted in differentiation of proteins from glycoproteins. More recently, an array composed of different cationic functionalized gold nanoparticles conjugated with an anionic fluorescent polymer poly(p-phenyleneethynylene) (PPE) has been created to detect and identify proteins7. The competitive binding between protein analytes and quenched PPE/gold nanoparticle complexes regenerated fluorescence, producing distinct recognition patterns for proteins. In this study, the functionalized nanoparticle-protein interactions were tuned by varying physicochemical properties of nanoparticle end groups. Furthermore, it was shown that this approach is effective for protein analysis in complex and protein-rich medium such as human serum at physiologically relevant concentrations, thus showing the potential of eT in profiling real samples for diagnosing disease states8.
Though very promising, these systems have some inherent limitations. They require designing and synthesizing from 5 to 29 CRRs with quite complicated structures. In addition, unlike the olfactory system that is reset following each measurement, protein sensing requires preparing an array per sample. Finally, monitoring real-time binding events are extremely difficult.
In this context, a combinatorial approach was proposed by using a small number of simple and easily accessible molecules with different physicochemical properties (hydrophilic, hydrophobic, positively charged, negatively charged, neutral, etc.) as building blocks (BBs)9. By mixing BBs in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for binding protein was created. Notably, the self-assembled monolayers on this system allow easy tuning of a range of surface properties in a highly divergent fashion, enabling diverse combinatorial cross-reactive receptors (CoCRRs) to be rapidly and efficiently produced. Protein sensing was performed using an optical detection system, surface plasmon resonance imaging (SPRi). Briefly, a broad-beam monochromatic polarized light from a LED illuminates the whole CoCRR array area on the surface of the prism. A high resolution CCD video camera provides real-time difference images across all the spots of the CoCRR array. It captures all of the local changes at the surface of the CoCRR array providing detailed information on binding events and kinetic processes10. Meanwhile, with the help of imaging software, SPR images corresponding to spots are automatically converted to variations of reflectivity versus time, generating a series of kinetic binding curves called sensorgrams. Thus, SPRi allows a label-free, synchronous, parallel, and real-time observation of binding events. Additionally, the obtained CoCRR array is regenerable and reusable for protein analysis.
This protocol describes the construction of the electronic tongue by using only two small molecules as building blocks and illustrates its application for analysis of common proteins based on continuous recognition patterns obtained with SPRi.

<table><tbody><tr><td>SPRi apparatus&amp;nbsp;</td><td>Horiba Scientific-GenOptics</td><td></td><td>SPRi apparatus is placed in a temperature regulated incubator at 25 &amp;deg;C.</td></tr><tr><td>Incubator</td><td>Memmert</td><td></td><td></td></tr><tr><td>Syringe pump&amp;nbsp;</td><td>Cavro scientific instruments</td><td>Cavro XLP 6000</td><td></td></tr><tr><td>Micro Elite Degasser&amp;nbsp;</td><td>Alltech</td><td>AT590507</td><td></td></tr><tr><td>6-port medium pressure injection valve</td><td>Upchurch Scientific</td><td></td><td>The volume of injection loop used is 500 &amp;micro;l.</td></tr><tr><td>Femto plasma cleaner (version 7)</td><td>Diener Electronic</td><td></td><td>On-line degassing system with 2 channel.</td></tr><tr><td>Spotter</td><td>Siliflow</td><td></td><td>It is a non-contact piezoelectric spotter.</td></tr><tr><td>SPRi-Biochip</td><td>Horiba Scientific-GenOptics</td><td>36000067</td><td>The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.</td></tr><tr><td>Erythrina cristagalli lectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L5390</td><td></td></tr><tr><td>Arachis hypogaea lectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L0881</td><td></td></tr><tr><td>Myoglobin</td><td>Sigma-Aldrich</td><td>M1882</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>CXCL12&amp;alpha;</td><td></td><td></td><td>Provided by Dr. Hugues Lortat-Jacob; for preparation details, see supporting information in reference 9</td></tr><tr><td>CXCL12&amp;gamma;&amp;nbsp;</td><td></td><td></td><td>Provided by Dr. Hugues Lortat-Jacob; for preparation details, see supporting information in reference 9</td></tr><tr><td>Lactose</td><td></td><td></td><td>Provided by Prof. David Bonnaff&amp;eacute;; for preparation details, see supporting information in reference 9</td></tr><tr><td>Sulfated lactose</td><td></td><td></td><td>Provided by Prof. David Bonnaff&amp;eacute;; for preparation details, see supporting information in reference 9</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5150</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>274348</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>S0751</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr></tbody></table>

electronic tongue generating continuous recognition patterns for protein analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

To probe the ability of the electronic tongue for common protein analysis, three proteins were used: AHL, myoglobin and lysozyme. For each protein, a distinct 2D continuous evolution profile, CEP, was generated by the eT, as shown in Figure 6.
In addition, thanks to SPRi, which is able to monitor the real-time adsorption and desorption kinetics, for each protein a time dependent continuous recognition pattern, called 3D continuous evolution landscape (CEL), was generated. In <...

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Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

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Research

JoVE Journal

Bioengineering

7.8K Views.  Institut Nanosciences et Cryogénie, CEA-Grenoble. A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination. 

Video: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Automated Lectin-Based Glycan Profiling Using Bead Array in a Single Tip (BIST)

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

Author Spotlight: Advancing Protein Glycosylation Research Using a Fully Automated System

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

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