The authors thank Heedeok Hong's group at the Department of Chemistry, Michigan State University, for kindly providing the Escherichia coli cells for the experiments. The authors thank the support from the National Institute of General Medical Sciences, the National Institutes of Health (NIH) through Grant R01GM118470 (to X. Liu) and Grant R01GM125991 (to L. Sun and X. Liu).

<ol>
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The authors have nothing to disclose.

Here we provide a detailed protocol to use CZE-MS/MS forthe high-resolution characterization of proteoforms in simple protein samples and for the large-scale identification of proteoforms in complex proteome samples. A diagram of the CZE-ESI-MS/MS system is shown in Figure 1. There are four critical steps in the protocol. First, the preparation of high-quality LPA coating on the inner wall of the separation capillary is extremely important. An LPA-coated separation capillary can reduce the E...

Top-down proteomics (TDP) aims for the large-scale characterization of proteoforms within a proteome. TDP relies on the effective liquid-phase separation of intact proteins before electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analysis due to the high complexity and large concentration dynamic range of the proteome1,2,3,4,5. Capillary zone electrophoresis (CZE) is a powerful technique for the separation of biomolecules based on their size-to-charge ratios6. CZE is relatively simple, requiring only an open tubular-fused silica capillary, a background electrolyte (BGE), and a power supply. A sample of intact proteins can be loaded into the capillary using pressure or voltage, and separation is initiated by immersing both ends of the capillary in the BGE and applying a high voltage. CZE can approach ultra-high separation efficiency (&#62; one million theoretical plates) for the separation of biomolecules7. CZE-MS has a drastically higher sensitivity than widely used reversed-phase liquid chromatography (RPLC)-MS for the analysis of intact proteins8. Although CZE-MS has a great potential for large-scale top-down proteomics, its wide application in proteomics has been impeded by several issues, including a low sample-loading capacity and narrow separation window. The typical sample loading volume in CZE is about 1% of the total capillary volume, which usually corresponds to less than 100 nL9,10,11. The separation window of CZE is usually less than 30 min due to the strong electroosmotic flow (EOF)9,10. These issues limit the CZE-MS/MS for the identification of a large number of proteoforms and low abundant proteoforms from a complex proteome.
Much effort has been made to improve the sample loading volume of CZE via online sample concentration methods (e.g., solid-phase microextraction [SPME]12,13, field-enhanced sample stacking [FESS]9,11,14, and dynamic pH junction15,16,17,18). FESS and dynamic pH junction are simpler than SPME, only requiring a significant difference between the sample buffer and the BGE in conductivity and pH. FESS employs a sample buffer with much lower conductivity than the BGE, leading to a stacking of analytes on the boundary between the sample zone and the BGE zone in the capillary. Dynamic pH junction utilizes a basic sample plug (e.g., 50 mM ammonium bicarbonate, pH 8) and an acidic BGE (e.g., 5% [v/v] acetic acid, pH 2.4) on both sides of the sample plug. Upon application of a high positive voltage at the injection end of the capillary, titration of the basic sample plug occurs, focusing the analytes into a tight plug before undergoing a CZE separation. Recently, the Sun group systematically compared FESS and dynamic pH junction for the online stacking of intact proteins, demonstrating that dynamic pH junction could produce much better performance than FESS for the online concentration of intact proteins when the sample injection volume was 25% of the total capillary volume19.
Neutrally coated separation capillaries (e.g., linear polyacrylamide [LPA]) have been employed to reduce the EOF in the capillary, slowing down the CZE separation and widening the separation window20,21. Recently, the Dovichi group developed a simple procedure for the preparation of stable LPA coating on the inner wall of capillaries, utilizing ammonium persulfate (APS) as the initiator and temperature (50 &#176;C) for free radical production and polymerization22. Very recently, the Sun group employed the LPA-coated separation capillary and the dynamic pH junction method for the CZE separation of intact proteins, reaching a microliter-scale sample loading volume and a 90-min separation window19. This CZE system opens the door to using CZE-MS/MS for large-scale top-down proteomics.
CZE-MS requires a highly robust and sensitive interface to couple CZE to MS. Three CE-MS interfaces have been well developed and commercialized in the history of CE-MS, and they are the co-axial sheath-flow interface23, the sheathless interface using a porous tip as the ESI emitter24, and the electro-kinetically pumped sheath flow interface25,26. The electro-kinetically pumped sheath-flow-interface-based CZE-MS/MS has reached a low zeptomole peptide detection limit9, over 10,000 peptide identifications (IDs) from the HeLa cell proteome in a single run14, a fast characterization of intact proteins11, and highly stable and reproducible analyses of biomolecules26. Recently, the LPA-coated separation capillary, the dynamic pH junction method, and the electro-kinetically pumped sheath flow interface were used for large-scale top-down proteomics of an Escherichia coli (E. coli) proteome19,27. The CZE-MS/MS platform approached over 500 proteoform IDs in a single run19 and nearly 6,000 proteoform IDs via coupling with size-exclusion chromatography (SEC)-RPLC fractionation27. The results clearly show the capability of CZE-MS/MS for large-scale top-down proteomics.
Herein, a detailed procedure of using CZE-MS/MS for large-scale top-down proteomics is described. The CZE-MS/MS system employs the LPA-coated capillary to reduce the EOF in the capillary, the dynamic pH junction method for the online concentration of proteins, the electro-kinetically pumped sheath flow interface for coupling CZE to MS, an orbitrap mass spectrometer for the collection of MS and MS/MS spectra of proteins, and a TopPIC (TOP-Down Mass Spectrometry-Based Proteoform Identification and Characterization) software for proteoform ID via database search.

<table><tbody><tr><td>Fused silica capillary</td><td>Polymicro Technologies</td><td>1068150017</td><td>50 &amp;micro;m i.d. 360 &amp;micro;m o.d.</td></tr><tr><td>Sodium hydroxide pellets</td><td>Macron Fine Chemicals</td><td>7708-10</td><td>Corrosive</td></tr><tr><td>LC-MS grade water</td><td>Fisher Scientific</td><td>W6-1</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>SA48-1</td><td>Corrosive</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A456-4</td><td>Toxic, Health Hazard</td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Sigma-Aldrich</td><td>M6514</td><td>Moisture and heat sensitive</td></tr><tr><td>Hydrofluoric acid</td><td>Acros Organics</td><td>423805000</td><td>Highy toxic</td></tr><tr><td>Acrylamide</td><td>Acros Organics</td><td>164855000</td><td>Toxic, health hazard</td></tr><tr><td>Ammonium persulfate</td><td>Sigma-Aldrich</td><td>A3678</td><td>Health hazard, Oxidizer</td></tr><tr><td>lysozyme</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>Cytochrome C</td><td>Sigma-Aldrich</td><td>C7752</td><td></td></tr><tr><td>Myoglobin</td><td>Sigma-Aldrich</td><td>M1882</td><td></td></tr><tr><td>&amp;szlig;-casein</td><td>Sgma-Aldrich</td><td>C6905</td><td></td></tr><tr><td>Carbonic anhydrase</td><td>Sigma-Aldrich</td><td>C3934</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Urea</td><td>Alfa Aesar</td><td>36428-36</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td>Health Hazard</td></tr><tr><td>Iodoacetamide</td><td>Fisher Scientific</td><td>AC122270250</td><td>Health Hazard</td></tr><tr><td>Formic Acid</td><td>Fisher Scientific</td><td>A117-50</td><td>Corrosive, Health Hazard</td></tr><tr><td>C4 trap column</td><td>Sepax Technologies</td><td>110043-4001C</td><td>3 &amp;micro;m particles, 300 &amp;Aring; pores, 4.0 mm i.d. 10 mm long</td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>A998SK-4</td><td>Toxic, Oxidizer</td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma-Aldrich</td><td>1066-33-7</td><td></td></tr><tr><td>Nalgene rapid-flow filters</td><td>Thermo Scientific</td><td>126-0020</td><td>0.2 &amp;micro;m CN membrane, and 50 mm diameter</td></tr><tr><td>E. coli cells</td><td></td><td></td><td>K-12 MG1655</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>BCA assay</td><td>Thermo Scientific</td><td>23250</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A11-1</td><td></td></tr><tr><td>HPLC system for protein desalting</td><td>Agilient</td><td>1260 Infinity II</td><td></td></tr><tr><td>Acetic Acid</td><td>Fisher Scientific</td><td>A38-212</td><td></td></tr><tr><td>CE autosampler</td><td>CMP Scientific</td><td>ECE-001</td><td></td></tr><tr><td>Electro-kinetically pumped sheath flow interface</td><td>CMP Scientific</td><td></td><td></td></tr><tr><td>Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Sutter flaming/brown micropipette puller</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>Ultrasonic cell disruptor for cell lysis</td><td>Branson</td><td>101063196</td><td>Model S-250A</td></tr><tr><td>Vaccum concentrator</td><td>Thermo Fisher Scientific</td><td>SPD131DDA-115</td><td></td></tr></tbody></table>

large-scale top-down proteomics using capillary zone electrophoresis tandem mass spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

Figure 1&#160;shows a diagram of the dynamic pH-junction-based CZE-ESI-MS system used in the experiment. A long plug of the sample in a basic buffer is injected into an LPA-coated separation capillary filled with an acidic BGE. After applying high voltages I and II, the analytes in the sample zone will be concentrated via the dynamic pH junction method. To evaluate the performance of the CZE-MS system, a standard protein mixture (cytochrome c, lysozy...

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Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

large-scale-top-down-proteomics-using-capillary-zone-electrophoresis

Research

JoVE Journal

Chemistry

9.4K Views.  Michigan State University. Deze methode kan helpen bij het verbeteren van grootschalige en hoge resolutie karakterisering van eiwitisovormen en eiwit post-translationele wijzigingen in cellen. Het belangrijkste voordeel van deze techniek is dat capillaire zone elektroforese massaspectrometrie biedt zeer efficiënte scheiding en zeer gevoelige detectie van intacte eiwitten. Om de capillaire voor te behandelen, droogt u het minstens 12 uur met stikstofgas op 10 psi.Vul vervolgens de capillaire met 50% volume-per-v...

Video: Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Biochemistry

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) ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

Immunology and Infection

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Biology

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

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