Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.
22 Related JoVE Articles!
Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration
Institutions: University of Edinburgh.
Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2
. Neural crest cell migration is extensively studied in vitro
but in vivo
methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo
. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6
. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo
. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR
reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.
Developmental Biology, Issue 87, mouse, melanoblast, skin, confocal microscopy, air-liquid interface
Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays
Institutions: University of Chicago.
tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli
and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli
and S. cerevisiae
have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae
. While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli
and human cell cultures[7, 8].
Cellular Biology, Issue 40, tRNA, aminoacylation, charging, microarray, S. cerevisiae
Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection
Institutions: Florida International University.
Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+
binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (τ < 20 μsec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.
Chemistry, Issue 84, photothermal techniques, photothermal beam deflection, volume change, enthalpy change, calcium sensors, potassium channel interaction protein, DM-nitrophen
Dissection of Saccharomyces Cerevisiae Asci
Institutions: Concordia University.
Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae
exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.
Cellular Biology, Issue 27, asci, ascospores, diploid, zygote, sporulation, yeast dissection, micromanipulator
Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry
Institutions: Virginia Polytechnic Institute and State University.
siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N
. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to A
f SidA, is reduced by NADPH2,3
. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1
), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in A
f SidA by NADPH is directly measured. We then use double mixing settings where A
f SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1
). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4
. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6
Bioengineering, Issue 61, Stopped-flow, kinetic mechanism, SidA, C4a-hydroperoxyflavin, monooxygenase, Aspergillus fumigatus
Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development
Institutions: Consiglio Nazionale delle Ricerche.
The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.
Chemistry, Issue 74, Biochemistry, Chemical Engineering, Chemical Biology, chemical analysis techniques, chemistry (general), life sciences, radiation effects (biological, animal and plant), biomarker, biomimetic chemistry, free radicals, trans lipids, cyclopurine lesions, DNA, chromatography, spectroscopy, synthesis
Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Institutions: The Hebrew University of Jerusalem, The Hebrew University of Jerusalem.
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
Chemistry, Issue 82, solution structure determination, NMR, peptide models, copper-binding proteins, copper complexes
Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides (CHIPS)
Institutions: Max-Planck-Institut fuer Kohlenforschung.
The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order to streamline synthesis by saving steps, time and material compared to conventional methods that require the introduction and removal of activating or directing groups. Therefore, the functionalization of C-H bonds is also attractive for green chemistry. Under oxidative conditions, two C-H bonds or one C-H and one heteroatom-H bond can be transformed to C-C and C-heteroatom bonds, respectively. Often these oxidative coupling reactions require synthetic oxidants, expensive catalysts or high temperatures. Here, we describe a two-step procedure to functionalize indole derivatives, more specifically tetrahydrocarbazoles, by C-H amination using only elemental oxygen as oxidant. The reaction uses the principle of C-H functionalization via Intermediate PeroxideS (CHIPS). In the first step, a hydroperoxide is generated oxidatively using visible light, a photosensitizer and elemental oxygen. In the second step, the N-nucleophile, an aniline, is introduced by Brønsted-acid catalyzed activation of the hydroperoxide leaving group. The products of the first and second step often precipitate and can be conveniently filtered off. The synthesis of a biologically active compound is shown.
Chemistry, Issue 88, Catalysis, Photocatalysis, C-H functionalization, Oxygen, Peroxides, Indoles, Pharmaceuticals
Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Institutions: University of Colorado Anschutz Medical Campus, University Hospital of Essen.
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo
using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo
pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo
using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo
bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro
and in vivo
and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Medicine, Issue 79, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
Sigma's Non-specific Protease Activity Assay - Casein as a Substrate
Institutions: Sigma Aldrich.
Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, click here
biochemistry, Issue 19, protease, casein, quality control assay, folin and ciocalteu's reagent, folin's reagent, colorimetric detection, spectrophotometer, Sigma-Aldrich
Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups
Institutions: University of Notre Dame .
Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2
Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n-
(E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6
Notable milestones are the oxidative coupling of Ge94-
clusters to oligomers and infinite chains,7-19
capping by transition-metal organometallic fragments,26-34
insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47
addition of main-group organometallic fragments as exo-bonded substituents,48-50
and functionalization with various organic residues by reactions with organic halides and alkynes.51-58
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4
from which the Ge94-
clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4
is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3
. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2
. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2
]•en which were characterized by single-crystal X-ray diffraction.
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.
Biochemistry, Issue 60, Zintl ions, deltahedral clusters, germanium, intermetallics, alkali metals
Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells
Institutions: Columbia University, Columbia University.
Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the Fo
-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.
Bioengineering, Issue 77, Microbiology, Cellular Biology, Molecular Biology, Biochemistry, life sciences, roGFP, redox-sensitive green fluorescent protein, GO-ATeam, ATP, FRET, ROS, mitochondria, biosensors, GFP, ImageJ, microscopy, confocal microscopy, cell, imaging
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
Institutions: Charité - Universitätsmedizin Berlin, Free University Berlin, Charité - Universitätsmedizin Berlin, Charité - Universitätsmedizin Berlin.
Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+
CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+
CSCs and CD133-
bulk, which can be cultivated and functionally analyzed thereafter.
Cancer Biology, Issue 73, Medicine, Stem Cell Biology, Cellular Biology, Molecular Biology, Biomedical Engineering, Genetics, Oncology, Primary cell culture, melanoma, MACS, cancer stem cells, CD133, cancer, prostate cancer cells, melanoma, stem cells, cell culture, personalized treatment
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Institutions: University of Heidelberg.
All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1
H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g.
crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex.
Chemistry, Issue 81, Molecular Chaperones, mass spectrometers, Amino Acids, Peptides, Proteins, Enzymes, Coenzymes, Protein dynamics, conformational changes, allostery, protein folding, secondary structure, mass spectrometry
A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry
Institutions: University of Münster, Carnegie Institution for Science.
The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii
, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14
N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f
). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g.
used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.
Microbiology, Issue 85, Sucrose density gradients, Chlamydomonas, multiprotein complexes, 15N metabolic labeling, thylakoids
Determination of the Gas-phase Acidities of Oligopeptides
Institutions: University of the Pacific.
Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6
. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8
, the results are often complicated by solvent effects 7
. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10
. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3
CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1
. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2
). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.
Chemistry, Issue 76, Biochemistry, Molecular Biology, Oligopeptide, gas-phase acidity, kinetic method, collision-induced dissociation, triple-quadrupole mass spectrometry, oligopeptides, peptides, mass spectrometry, MS
In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries
Institutions: University of Sydney, University of Wollongong, Australian Synchrotron, Australian Nuclear Science and Technology Organisation, University of Wollongong, University of New South Wales.
Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2
However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3
This research is challenging, and we outline a method to address these challenges using in situ
NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries.
We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the ‘roll-over’ cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ
NPD experiment and initial directions are presented on how to analyze such complex in situ
Physics, Issue 93, In operando, structure-property relationships, electrochemical cycling, electrochemical cells, crystallography, battery performance
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae
Institutions: VU University Amsterdam.
In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo
, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871
, who showed that it was possible to reconstitute these complexes in vitro
starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro
reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g.,
pigment binding sites) or protein domain (e.g.,
protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro
currently used in our laboratory,
and examples describing applications of the method are provided.
Biochemistry, Issue 92, Reconstitution, Photosynthesis, Chlorophyll, Carotenoids, Light Harvesting Protein, Chlamydomonas reinhardtii, Arabidopsis thaliana
Synthesis and Characterization of Functionalized Metal-organic Frameworks
Institutions: Northwestern University, Warsaw University of Technology, King Abdulaziz University.
Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g.
, other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2
drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.
Chemistry, Issue 91, Metal-organic frameworks, porous coordination polymers, supercritical CO2 activation, crystallography, solvothermal, sorption, solvent-assisted linker exchange
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
Institutions: Scientific Instruments.
Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF · (OD260-OD320)· NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1
Protein concentration results can be expressed as μg/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.
Molecular Biology, Issue 48, Nucleic acid quantitation, protein quantitation, micro-volume analysis, label quantitation