Name: time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics
Uploaded: 2017-04-17T12:30:00
Description: Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

We gratefully acknowledge Dr. Markus Zweckstetter for providing the pdb coordinate file for the 'native' tau ensemble predicted from his NMR work, with contributed analysis tools provided by Dr. Adnan Sljoka. Funding for this work was provided by the Natural Science and Engineering Research Council of Canada (NSERC) ENGAGE Grant program.

NOTE: Please consult all relevant material safety data sheets (MSDS) before use. Fumes produced by laser ablation of poly(methyl methacrylate) (PMMA) can be toxic. Be sure that the laser engraver is connected to a working ventilation system. Use all appropriate safety practices when building the microfluidic device including the use of engineering controls (fume hood, sharps container) and personal protective equipment (safety glasses, face mask, gloves, lab coat, full length pants, closed-toe shoes). It is of utmost imp...

<ol>
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While structural biology methods such as X-ray crystallography and NMR are advantageous because they provide extremely detailed structures of proteins, these pictures are often static. The characterization of transient species and weakly structured domains continues to be elusive when studied by these conventional methods. Therefore, in order to gain dynamic insights on these types of systems it is important to work at rapid time scales. We have successfully applied TRESI-HDX-MS to obtain detailed insights on the conform...

Over the past several decades, significant advancements have been made in the development of analytical techniques designed to measure protein structure and dynamics1,2,3,4. While X-ray crystallography remains the principle tool for determining protein structure, high concentrations of protein are needed and extensive optimization is required to produce diffraction quality crystals. Proteins that are difficult to crystallize, such as membrane-associated and intrinsically disordered proteins have classically been studied by hydrogen-deuterium exchange (HDX) NMR5. However, in recent decades, coupling of electrospray ionization mass spectrometry (ESI-MS) to HDX has rapidly gained popularity6,7.
Mass spectrometry offers a solution to many of the restrictions posed by X-ray crystallography and NMR. In particular, MS is highly sensitive (nM to &#181;M concentrations required), and there is virtually no limit on protein size. In addition, the high duty cycle of MS analysis allows for the possibility of studying proteins as they undergo enzymatic turnover, misfolding, complexation and other biologically-relevant processes. These processes often occur on the millisecond to second time scale and require rapid mixing of reagents prior to analysis.
The development of Time-Resolved ElectroSpray Ionization (TRESI) by Wilson and Konermann in 2003 allowed reactions to be monitored in pseudo-real time by ESI-MS. Their setup incorporated a capillary mixer with a continuously adjustable reaction chamber volume8. The device consists of two concentric capillaries, with the inner capillary sealed and a notch cut into its side to allow for mixing within the narrow inter-capillary space from the notch to the end of the inner capillary (typically 2 mm). When applied to HDX experiments, the inner capillary carries the protein of interest, the outer capillary carries the labeling D2O solution, which then undergoes mixing with the protein before entering the adjustable reaction chamber allowing for HDX labelling prior to direct transfer into the ESI source.
Briefly, HDX relies on backbone amide hydrogens undergoing exchange with deuterium atoms in solution9,10. The exchange is base-catalyzed at physiological pH, with acid-catalysis becoming prevalent at pH below approximately 2.6. The rate of exchange is based on four main factors: pH, temperature, solvent accessibility and intramolecular hydrogen bonding. As the former two factors are kept constant throughout the experiment, the rate of exchange, particularly at peptide backbone amide positions, is primarily dependent on protein structure11. Tightly folded regions with extensive, stable hydrogen bonding networks in &#945;-helices and &#946;-sheets will take up deuterium at substantially slower rates compared to loops and disordered regions (and sometimes not at all)12. This allows for global protein analysis, where perturbations in structure (e.g., upon aggregation or substrate binding) lead to differing deuterium uptake (Figure 1).
The kinetic capillary mixer can be incorporated into a microfluidic platform containing a proteolytic chamber for localization of the deuterium uptake. This proteolytic chamber is held at low pH in order to effectively quench the exchange reaction, and requires an immobilized acid protease in order to digest the protein into localized peptides (Figure 2). Monitoring backbone exchange at millisecond to second time scales is especially important for the characterization of conformational changes within difficult to characterize loop regions, molten globules, and intrinsically disordered proteins (IDPs)13,14. Alternatively, TRESI-HDX can also be used to characterize proteins that currently do not have a solved atomic structure through the methods of X-ray crystallography and NMR, using deuterium exchange coupled to the COREX algorithm (DX-COREX) approach15,16. This detailed protocol will apply TRESI-HDX to study tau, an IDP, in both it&#39;s native form as well as it&#39;s pathogenic hyperphosphorylated state. While native tau is one of the most well studied IDPs, little is known about its amyloidogenic counterpart13.

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time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry for studying protein structure and dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Digestion profiles of native and phospho-tau were similar, yielding a sequence coverage of 77.1 and 71.7% respectively. Deuterium uptake values of each peptide was determined by fitting the observed isotopic distributions with the theoretical distributions generated using an in-house developed FORTRAN software. The best fitting distributions are shown (Figure 3a) along with the associated deuterium uptake values. Uptake kinetic profiles are then generated, and were well d...

Watch this Scientific Journal Video about Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics at JoVE.com

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Conformational flexibility plays a critical role in protein function and the overall goal of Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry is to probe rapid structural changes that drive function in ordered and disordered proteins. This method can help answer key questions in the structural biology field, such as the craterization of transient protein confirmations, that's not a minimal to classical high resolution techniques such as electrocystography and NMR. The main advantage of this technique is that reactions can be monitored on the millisecond time scale, which is important for characterizing loop ridges, Moltinglobyals, and intrinsically disordered proteins.The implications of this technique extend toward therapy of neurodegenerative disorders. As they often involve the misfolding, or aggregation of intrinsically disordered protein regions. Though this method can provide insight into weekly structured proteins, it can also be applied to other systems, such as enzymes undergoing catalytic turnover, or the characterization of protein, protein and protein, ligan, interactions.To begin this procedure, laser a plate through an input channel for introducing the reagents, a pro dialysis chamber and an output channel, on a standard PMM8 block, using a laser engraver. Following this, cut a 30 gauge stainless steel metal capillary into two 10 centimeter pieces using a rotary tool for the 164th inch thick cut off disk. Use sand paper to smooth the end of the capillaries, which can be facilitated by viewing under a light microscope.Now, melt that capillaries into the etched PMM8 block using a sauntering iron. To construct a continuous flow, time resolved kinetic mixer, insert a 40 centimeter fused cilica glass capillary into a 28 gauge, steel, metal capillary of approximately 15 centimeters. Next, produce a two millimeter notch, using low power laser engraver settings on one end of the inner glass capillary and seal this end of the inner glass capillary.This is a critical step that ensures orthogonal and efficient mixing of the protein, which exits through the notch which an on common deterior. The two millimeter inter capillary space between the notch and the end of the capillary is the mix and volume. Attach appropriate size fittings and tubing sleeves and incorporate a T connector, which will be used to deliver deterium.Next, line up the inner glass capillary with the end of the metal capillary which can be facilitated by viewing under a light microscope. Mark this as the zero millimeter position. Attach this kinetic mixer to one end of the mixing T.Then attach a 40 centimeter fused silica glass capillary to the opposite end of the kinetic mixer on the mixing T.For pepsin activation, suspend 20 milligrams of pepsin from Porsena gassic mucosa in one milliliter of coupling buffer.Add 50 milligrams of NHS activated agro speeds to the resuspended protiens and rotate gently over night, at four degrees Celsius. On the following day, spin down the mixture at one thousand times G for two minutes at room temperature to collect the resin. Then aspirate the unbound protiyes.Following this, incubate the agros in one milliliter of blocking buffer and rotate gently at roomtemperature for one hour. After collecting the resin and aspirating the blocking buffer, incubate the pepsin agguros with one milliliter of five percent acedic acid for five minutes. Spin down the mixture at 1, 000 times G, for two minutes at room temperature, to collect the resin, then aspirate the super natent.After repeating the previous steps, for a total of three washes, store the beads in one milliliter of acedic acid at four degrees Celsius for longterm use. To assemble the device, fill a protialysis chamber with a slurry of activated pepsin agguros beads in five percent acedic acid using a sterile spatula. Place the PMMA micro falitic platform in between two blank PMMA blocks as a cover to seal the device, lined with silicone rubber to create a liquid tight seal.Use metallic clamping plates to pressure seal the device. Then attach the mixing T, to the inlet of the device using appropriate fittings. Now flow five percent acedic acid thought the acid line at a rate of ten microliters a minute.Then flow 50 millimeter amonia acid buffer through the protein line at a rate of one microliter per minute. Couple the device to the front end of a modified, quadrapole time of flight, or Qtof mass spectrometer, using and adjustable insulated stage to achieve optimal electro spray conditons. After setting the ESI-MS conditions, acquire a pepsin only spectrum.Next, introduce 50, to 100 micromoler of tau, or phospho-tau protein at a rate of one microliter per minute, where the 50 millimeter amonium acitate buffer was previously flowing. After allowing the system to equilibrate for at least 10 minutes, before acquisition, acquire protein only spectrum. While the Tao, or phospho tao protein is flowing, introduce duteriumoxide at a rate of three microliters per minute, via the T connector, and allow to react in the kinetic mixer.In order to increase the labeling time, manually pull back the position of the inner glass capillary to achieve mixing times of 42 milliseconds to eight seconds, allowing the system to equilibrate for at least 10 seconds in between each pull back. This animation depicts the workflow for a typical experiment. Protein exits through the notch and mixes with the oncoming duterium.Backbone M8 hydrogens are exchanged during the allotted reaction time, which can be varied by pulling back the protein capillary. The reaction is then quenched and then digested on a micro fluidic platform containing an outlet ESI needle. The resulting spectrum contains digested protein.The percentage of deuterium uptake is calculated for individual peptides and then mapped onto the 3D protein structure. Digestion profiles of native and phospho-tau were similar, yielding a sequence coverage of 77.1 and 71.7 percent, respectively, the best fitting isotopic distributions and the associated deuterium uptake values for four simple peptides are shown here. Observed kinetic plot of percent to deuterium uptake, versus time, for each peptide are used to extract the observed experimental rate.The calculated random quale profiles are shown for comparison and are used to extract the theoretical intrinsic grade. As expected, no protection factors were observed in the range, normally associated with secondary structure elements, confirming that native tau exhibits weak internal hydrogen bonding. Significant protection is observed at the N and C termini, the central domain, and the aggregation proned regions.In hyper phospho related tau, a general increase was observed in deuterium uptake across the protein. There are significant increases at the N and C termini and in the hexapeptide two region. Degree of protection factors are colored as a rainbow scheme, as shown here.Once mastered, this technique can be done in a few hours, if performed properly. While attempting this procedure, it is important to use regions of HPC grade, or higher, to minimize contaminants as they can interfere with analysis. Following this procedure, other methods, such as covalent labeling, chemical cross linking, computational modeling and docustudies can be performed in order to match three dimensional protein structures, or confirm areas of protein protein and protein legin interaction.After its development, this technique paved the way for researchers in the field of structural biology, to explore intrinsically disordered proteins and domains, most common in nuerodegenerative disease. After watching this video, you should have a good understanding of how to use time resolved electro spray ionization, hydrogen deuterium exchange, mass spectometry, for the study of protein structure and dynamics.

time-resolved-electrospray-ionization-hydrogen-deuterium-exchange

Research

JoVE Journal

Biochemistry

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 ...

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass spectrometry (TX-MS), a method combining quaternary protein structure modeling and chemical cross-linking mass spectrometry, creates high-accuracy structure models using data obtained from complex, unfractionated samples. This removes one of the major obstacles to protein complex structure analysis because the proteins of interest no longer need to be purified in large quantities. Cheetah-MS web server was developed to make the simplified version of the protocol more accessible to the community. Considering the tandem MS/MS data, Cheetah-MS generates a Jupyter Notebook, a graphical report summarizing the most important analysis results. Extending the Jupyter Notebook can yield more in-depth insights and better understand the model and the mass spectrometry data supporting it. The technical protocol presented here demonstrates some of the most common extensions and explains what information can be obtained. It contains blocks to help analyze tandem MS/MS acquisition data and the overall impact of the detected XLs on the reported quaternary models. The result of such analyses can be applied to structural models that are embedded in the notebook using NGLView.

Targeted cross-linking mass spectrometry creates quaternary protein structure models using mass spectrometry data acquired using up to three different acquisition protocols. When executed as a simplified workflow on the Cheetah-MS web server, the results are reported in a Jupyter Notebook. Here, we demonstrate the technical aspects of how the Jupyter Notebook can be extended for a more in-depth analysis.

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...