This research was supported by Award Number R00RR024105 from the National Center For Research Resources, National Institute of Health.

1. Reductive Methylation of Lysozyme
<ol>
	<li>Prepare 500 ml of a 50 mM sodium phosphate buffer at pH 7.5. Store at room temperature.</li>
	<li>Label three 1 ml microcentrifuge tubes for fully labeled sample (FLS), low pH partially labeled sample (LpH), and high pH partially labeled sample (HpH).</li>
	<li>Weigh out 10 mg of lysozyme and dissolve in 2 ml of sodium phosphate buffer to create an aqueous solution with a concentration of 5 mg/ml.&#160;Cover the tubes with aluminum foil to protect the formaldehyde from degradation due light exposure. Add 500 &#956;l of lysozyme solution to each tube.</li>
	<li>Prepare fresh 1.0 M solutions of dimethylamine borane complex (DMAB) and 13C-formaldehyde. Store at 0 &#176;C.</li>
	<li>For the fully labeled sample, use a molar ratio of 1:10 (reactive amine: formaldehyde), and for the partially labeled samples, use a 1:5 ratio. Alternatively, the ratio can be optimized. For example, in order to see the dominance of the &#945;-13C-dimethylamine peak at low pH, a 2:7 ratio was required.</li>
	<li>Add 6.1 &#956;l of 1.0 M DMAB to FLS, add 3.2 &#956;l of 1.0 M DMAB to LpH and HpH, and gently vortex. Then add 12.3 &#956;l of 1.0 M 13C-formaldehyde to FLS and 6.1 &#956;l of 1.0 M 13C-formaldehyde to LpH and HpH. Shake the reaction mixtures at 4 &#176;C for 2 hr.</li>
	<li>Add another aliquot of 1.0 M DMAB and 13C-formaldehyde as in the previous step and shake the reaction mixture overnight at 4 &#176;C for a total reaction time of 18-24 hr.</li>
	<li>Exchange the sample&#39;s buffer to phosphate at pH 7.5 to remove excess reagents and side products using a centrifugal filter (3 kDa molecular weight cut-off).</li>
	<li>For the partially labeled samples, reductively methylate the samples at pH 7.5 with natural abundance formaldehyde to create a homogeneously modified protein. Prepare new 1.0 M solutions of DMAB and natural abundance formaldehyde.</li>
	<li>Cover the sample tubes with foil, add 6.1 &#956;l of DMAB to LpH and HpH, and gently vortex. Then add 12.3 &#956;l of natural abundance formaldehyde. Shake the reaction mixtures at 4 &#176;C for 2 hr.</li>
	<li>Add another aliquot of 1.0 M DMAB and natural abundance formaldehyde as in the previous step and shake the reaction mixtures overnight at 4 &#176;C for a total reaction time of 18-24 hr. Then buffer exchange using steps 2.5-2.6&#160;and store 50 &#956;g samples at -20 &#176;C for MS.</li>
</ol>
2. Prepare the Sample for NMR Analysis
<ol>
	<li>Prepare a 2.0 M sodium borate buffer solution at pH 8.5 in H2O.</li>
	<li>Add 375 &#956;l of 2.0 M sodium borate buffer solution to four 15 ml conical tubes. Parafilm the tops and poke holes. Place the flask in a lyophilizing flask and freeze the sample.</li>
	<li>Place the frozen sample on a lyophilizer to dry. Remove when dry.</li>
	<li>Using a pipette, add 15 ml of D2O to each tube and mix.</li>
	<li>Label three 4 ml centrifugal filters (3 kDa molecular weight cut-off) with FLS, LpH, and HpH. Add the corresponding sample to the filter tubes (~500 &#956;l). Add 3.0 ml of 50 mM sodium borate buffer at pH 8.5 to each tube. Make a balance tube for the third tube and concentrate the samples to 500 &#956;l using a centrifuge with a 35&#176; fixed angle rotor at 4 &#176;C and 7,500 rcf.</li>
	<li>Empty the trap under each filter. Add another 3.0 ml of buffer to the samples and concentrate the samples again to 500 &#956;l. Repeat step 2.5&#160;4x.</li>
	<li>Using the bicinchoninic acid (BCA) protein assay36, determine the concentration of each sample. Dilute the lysozyme samples with 50 mM sodium borate buffer to make 150 &#956;M samples.</li>
	<li>Transfer 530 &#956;l to a 5 mm NMR tube or 250 &#956;M to a 5 mm magnetic susceptibility matched tube.</li>
	<li>Prepare an 80 mM stock solution of 1,2-dichloroethane-13C2 in D2O. Store this stock solution at -80 &#176;C. Dilute to 24 mM in 80 &#956;l and transfer to coaxial insert tube to use as a reference in NMR analysis. Analyze the samples with NMR. Note: Run a 1D 1H-13C HSQC and integrate the peak areas for each resonance. Peak areas should be the same for fully labeled sample.</li>
</ol>
3. Aminopeptidase Degradation of Lysozyme
<ol>
	<li>Prepare a 5 mg/ml solution of lysozyme in ultrapure water.</li>
	<li>Add 250 &#956;l of the lysozyme solution to a clean, 1 ml centrifuge tube.</li>
	<li>Add 25 &#956;l of a 0.1 M tricine solution, pH 8.0, and 6 &#956;l of a 2.16 &#956;g/ml Aeromonas proteolytica aminopeptidase solution.</li>
	<li>Incubate at 37 &#176;C for 6 hr.</li>
	<li>Add an additional 6 &#956;l of the 2.16 &#956;g/ml aminopeptidase solution to the sample for a 1:50 molar ratio of aminopeptidase to lysozyme. Incubate for an addition 20 hr at 37 &#176;C.</li>
	<li>Desalt 30 &#956;l of the sample using a C18 spin column, (follow manufacturer&#39;s protocol). Store at -20 &#176;C for MALDI MS analysis.</li>
	<li>Prepare the remaining protein sample for NMR as described in protocol&#160;2).</li>
</ol>
4. Prepare Samples for Mass Spectrometry
<ol>
	<li>Using the 50 &#956;g of sample reserved in step 2.3, exchange the buffer to 100 mM ammonium bicarbonate, pH 8.0, and concentrate to approximately 50 &#956;l using a 1 ml centrifugal filter (3 kDa molecular weight cut-off). Transfer the sample to a 1 ml centrifuge tube and wrap in aluminum foil.</li>
	<li>Add 2.0 &#956;l of 500 mM dithiothreitol to reduce the disulfide bonds. Incubate the solution at 60 &#176;C for 1 hr.</li>
	<li>Add 2 &#956;l of freshly prepared 500 mM iodoacetamide to alkylate the thiol groups. Incubate at room temperature for 20 min.</li>
	<li>Remove the foil and add 1.0 &#956;l of the 500 mM dithiothreitol solution to quench the reaction.</li>
	<li>Add 2.0 &#956;g of sequencing grade trypsin to digest the protein. Incubate the mixture at 37 &#176;C for 16-24 hr.</li>
	<li>Add TFA until the pH is less than 2 to quench the digestion. (Formic and acetic acids can be used as well)</li>
	<li>Desalt the sample on a C18 spin column and elute the protein in 80% acetonitrile/0.1% TFA. Dry the sample using a vacuum concentrator.</li>
	<li>Reconstitute the sample in 5.0 &#956;l of 50% acetonitrile/0.1% TFA for MALDI MS analysis. Mix equal volumes of the reconstituted sample and a saturated solution of 2,5-dihydroxybenzoic acid matrix solution on the MALDI target plate. Analyze the samples on a MALDI MS. Note: Use tandem MS to confirm the identity of peptide peaks. Smooth the mass spectra and analyze. For each dimethylamine-containing peptide, determine the peak intensity and peak area of each isotope in the profile. Calculate the percentage of 13C-incorporation as described previously34.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+the+Aminopeptidase+from+Aeromonas+proteolytica+on+Mg2+/Al3++Layered+Double+Hydroxide+Particles.">Immobilization of the Aminopeptidase from Aeromonas proteolytica on Mg2+/Al3+ Layered Double Hydroxide Particles.</a> Acs Appl. Mater., &amp; Interfaces. 2 (10), 2828-2832 (2010).</li><li>Poncz, L., Dearborn, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Resistance+to+Tryptic+Hydrolysis+of+Peptide-Bonds+Adjacent+to+N-epsilon,N-Dimethyllysyl+Residues.">The Resistance to Tryptic Hydrolysis of Peptide-Bonds Adjacent to N-epsilon,N-Dimethyllysyl Residues.</a> J. Biol. Chem. 258 (3), 1844-1850 (1983).</li></ol>

We have nothing to disclose.

Assigning the NMR signals of reductively 13C-methylated proteins is necessary to fully utilize this isotopic labeling method. The use of MS to aid in the assignment of the NMR signals thru correlation of the partial 13C-incorporation data is a promising technique. The advantage of this technique over other assignment methods is that only the primary amino acid sequence is needed. The MS-assisted assignment method is limited when the N-terminal amino acid is a lysine because the 13</sup...

Nuclear magnetic resonance (NMR) spectroscopy is a valuable structure elucidation tool for proteins1. NMR spectroscopy can be used to determine the solution structure of a protein in its native state. To overcome the low natural abundance of stable magnetic isotopes, it is necessary to incorporate 13C and 15N into the protein of interest. The most common method employed is recombinant expression in a bacterial host2-3. However, two disadvantages of bacterial host over-expression are it cannot produce post-translational modifications and does not work for all proteins3-4. When bacterial expression is not a viable route for protein production, over-expression in nonbacterial hosts can be used, but isotopic labeling is difficult and expensive5. Alternative expression methods for incorporating 13C and 15N isotopes into proteins for NMR analysis include sparse labeling techniques using metabolic precursors for methyl labeling6 and single 13C, 15N amino acids7-9. A chemical approach to sparse labeling used herein is the well-established reductive 13C-methylation reaction (Figure 1), where the primary amino groups on a protein - the N-terminal &#945;-amine and the lysine, side chain &#949;-amines - are methylated. Once monomethylamines are formed, the amine readily undergoes methylation again, due to the higher pKa value, to form dimethylamines.
Reductive methylation was first introduced as a method to chemically modify proteins by Means and Feeney10. The advantages of this reaction are its broad applicability and mild reactions conditions at buffered, physiological pH and low temperatures11,12.&#160;In the presence of formaldehyde and a reducing agent, such as dimethylamine borane complex (DMAB), the lysine &#949;-amino groups and the N-terminal &#945;-amino group are selectively methylated to produce dimethylated amines. Although formaldehyde is known to cross-link proteins through the formation of methylene bridges, this process is blocked by the reducing agent13,14.
Reductive methylation has been successfully used to study proteins with both NMR and x-ray crystallography. Reductive methylation is used to facilitate the crystallization of otherwise intractable proteins15. Hen egg white lysozyme was the first protein crystallized in its dimethylated form. The root-mean square difference between the heavy atoms in the methylated and unmethylated lysozyme structures is 0.40 &#197;16. This comparison demonstrates that the protein structure can be maintained after reductive methylation, making the reaction a viable, labeling tool for structure elucidation.
By using 13C-labeled formaldehyde in the reductive methylation reaction, 13C-dimethylated amines are produced. The 13C-dimethylamines are NMR-detectable probes that have been widely used to study protein dynamics, structure, and function. NMR and reductive 13C-methylation have been used to study protein-ligand and protein-protein interactions for the &#946;2 adrenergic receptor17, ribonuclease A18, lysozyme19, fd gene 5 protein20, and cytochrome c21. Similarly, structural and functional properties have been studied of reductively 13C-methylated ribonuclease A22, lysozyme23, fd gene 5 protein24, Clostridium pasteurianum ferredoxin25, Fc fragment of IgG26, apolipoprotein A-I27,&#160;and MIP-1&#945;28 The dynamics of the 13C-dimethylamino groups have been studied on concanavalin A29-30 and calmodulin31.
Even though reductive 13C-methylation has been used widely to study proteins with NMR, the labeling method has always been limited by the difficulties of assigning the NMR resonances26. Most assignment strategies for reductively 13C-methylated proteins have relied on small numbers of sites,20,28 known structural properties11,19,23,26,31,32. or extensive genetic modifications33. None of these studies successfully assigned all the 13C-dimethylamine peaks except for the calmodulin study, where the peaks were assigned by site-directed mutagenesis of each lysine33. In the study of MIP-1&#945; dimer formation, the use of mass spectrometry (MS) to aid in the NMR assignment of reductively 13C-methylated amines was first reported28. Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) MS was used to identify the lysine at the interface of the dimer. The partially methylated lysines of human MIP-1&#945; tryptic peptides were identified with MS and correlated with the appearance of mono- and dimethylamine signals of the intact protein observed in 2D 1H-13C HSQC NMR spectra28. Our group expanded on the use of MS and presented an assignment method that requires no prior knowledge of the protein&#39;s structure or properties other than the amino acid sequence34. This method is applicable to most proteins. One exception is when a protein has an N-terminal lysine because the MS isotopic profile of the N-terminal lysine &#945;- and &#949;-13C-dimethylamines cannot be independently measured.
Here we present two methods, one chemical and one enzymatic, to identify the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine sites of an N-terminal lysine residue. The first method was inspired by C&#243;rdova et al.&#160;who used pH to control the selectivity of protein acetylation35. The reaction favors the N-terminal &#945;-amine at low pH and the side chain &#949;-amines at high pH, allowing the protein &#945;- and &#949;-amino groups to be distinguished, in their studies, with capillary electrophoresis35. We demonstrate how high and low pH is used to alter the labeling of protein amino groups using the reductive 13C-methylation reaction and to allow application of the MS-assisted assignment strategy. The second method to assign the N-terminal &#945;- and &#949;-13C-dimethylamines takes advantage of the selective removal of the N-terminal residue(s) using recombinant Aeromonas proteolytica aminopeptidase.

<table><tbody><tr><td>Acetonitrile</td><td>Sigma</td><td>34851</td><td></td></tr><tr><td>Amicon Ultra 4 ml centrifugal filter (3k MWCO)</td><td>Fisher Scientific</td><td>UFC800308 (8pk)</td><td></td></tr><tr><td>Aminopeptidase</td><td>Creative Biomart</td><td>Aminopeptidase-86A</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma</td><td>A6141</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Sigma</td><td>A5132</td><td></td></tr><tr><td>Borane-dimethylamine complex</td><td>Sigma</td><td>180238</td><td>Make fresh 1 M solution before use.</td></tr><tr><td>Boric acid</td><td>Sigma</td><td>B6768</td><td></td></tr><tr><td>Bovine pancreas insulin</td><td>Sigma</td><td>I5500</td><td></td></tr><tr><td>C18 Spin Columns</td><td>Thermo Scientific</td><td>89870</td><td></td></tr><tr><td>Deuterium oxide</td><td>Cambridge Isotope Laboratories</td><td>DLM-4-100</td><td></td></tr><tr><td>1,2-dichloroethane-&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>714321</td><td>80 mM stock solution stored at -80 &amp;deg;C. Used as reference in NMR spectra at 24 mM.</td></tr><tr><td>Formaldehyde (36.5% wt/wt)</td><td>Sigma</td><td>33220</td><td>Make fresh 1 M solution before use.</td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C-formaldehyde (99%)</td><td>Cambridge Isotope Laboratories</td><td>CLM-806-1</td><td>Make fresh 1 M solution before use.</td></tr><tr><td>Lysozyme (hen egg white)</td><td>Sigma</td><td>L6376</td><td></td></tr><tr><td>Sodium phosphate (dibasic heptahydrate)</td><td>Sigma</td><td>S9390</td><td></td></tr><tr><td>Sodium phosphate (monobasic)</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Tricine</td><td>Sigma</td><td>T0377</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma</td><td>302031</td><td></td></tr><tr><td>700 MHz Varian VNMRS with 5 mm HCN 5922 probe</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Bruker UltrafleXtreme MALDI TOF/TOF</td><td>Bruker</td><td></td><td></td></tr></tbody></table>

methods to identify the nmr resonances of the 13c-dimethyl n-terminal amine on reductively methylated proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Reductive Methylation and pH
A pH induced chemoselective reductive 13C-methylation reaction has been demonstrated on lysozyme. At low pH, the reaction prefers the N-terminal &#945;-amine over the side chain &#949;-amines and vice versa at high pH. In solution the protein&#39;s amino groups exist in equilibrium between the free amine and the conjugate acid, which is tunable with pH. The optimum pH range for this reaction depends on the reducing agent used. In this case, dimethy...

Watch this Scientific Journal Video about Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins at JoVE.com

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

methods-to-identify-nmr-resonances-13c-dimethyl-n-terminal-amine-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

6.2K ビュー.  Louisiana State University. Two methods for assigning the α- and ε-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

ビデオ: Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

本質的に無秩序なタンパク質の複数のリン酸化の同定のための核磁気共鳴分光法

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

生化学

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

安定同位体標識するための還元ジメチル化を用いた定量的プロテオミクス

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

化学

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

固体核磁気共鳴分光法による高分子アセンブリの原子スケール構造の研究

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

核磁気共鳴分光法によるアルギニンメチロームの探索

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

がん研究

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

15μs-ms タイムスケールにおけるタンパク質コンフォメーションダイナミクスの研究のためのN CPMG緩和分散

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

質量分析に基づくプロテオミクスアプローチによるグローバルで信頼性の高いタンパク質R-メチル化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the peptide biomarkers of disease or environmental stress, identifying neuropeptides, hormones, and bioactive intracellular peptides, discovering antimicrobial and nutraceutical peptides from protein hydrolysates, and can be used in studies to understand the proteolytic processes. The recent advance in sample preparation, separation methods, mass spectrometry techniques, and computational tools related to protein sequencing has contributed to the increase of the identified peptides number and peptidomes characterized. Peptidomic studies frequently analyze peptides that are naturally generated in cells. Here, a sample preparation protocol based on heat-inactivation is described, which eliminates protease activity, and extraction with mild conditions, so there is no peptide bonds cleavage. In addition, the relative quantitation of peptides using stable isotope labeling by reductive methylation of amines is also shown. This labeling method has some advantages as the reagents are commercially available, inexpensive compared to others, chemically stable, and allows the analysis of up to five samples in a single LC-MS run.

This article describes a sample preparation method based on heat-inactivation to preserve endogenous peptides avoiding degradation post-mortem, followed by relative quantitation using isotopic labeling plus LC-MS.

ペプチド学研究におけるアミンの還元メチル化を用いたサンプル調製と相対定量

Peptidomics can be defined as the qualitative and quantitative analysis of peptides in a biological sample. Its main applications include identifying the ...

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

天然的に無秩序なタンパク質の自己会合の検出と特性評価のための常磁性緩和増強

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

High-Resolution Studies of Proteins in Natural Membranes by Solid-State NMR

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-(1H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

This work details robust basic routines on how to prepare isotope-labeled membrane protein samples and analyze them at high-resolution with modern solid-state NMR spectroscopy methods.

Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy ...

Nuclear Magnetic Resonance (NMR) Spectroscopy

Source: Laboratory of Dr. Henrik Sund&#233;n &#8211; Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis technique for organic chemists. With the help of NMR, the work in the organic lab has been facilitated tremendously. Not only can it provide information about the structure of a molecule but also determine the content and purity of a sample. Compared with other commonly encountered techniques for organic chemists &#8212; such as thermal analysis and mass spectrometry (MS) &#8212; NMR is a non-destructive method that is valuable when recovery of the sample is important.
One of the most frequently used NMR techniques for an organic chemist is proton (1H) NMR. The protons present in a molecule will behave differently depending on its surrounding chemical environment, making it possible to elucidate its structure. Moreover, it is possible to monitor the completion of a reaction by comparing NMR spectra of the starting material to that of the final product.
This video exemplifies how NMR spectroscopy can be used in the everyday work of an organic chemist. The following will be shown: i) preparation of an NMR sample. ii) Using 1H NMR to monitor a reaction. iii) Identifying the product obtained from a reaction with 1H NMR. The reaction that will be shown is the synthesis of an E-chalcone (3) from an aldehyde (1) and a ketone (2) (Scheme 1).1
<img alt="Scheme 1" src="/files/ftp_upload/5680/5680scheme1.jpg" /> 
Scheme 1. Synthesis of (2E)-3-(4-methoxyphenyl)-1-(4-methylphenyl)-2-propen-1-one.

NMR Spectroscopy: Principle, NMR Active Nuclei, Applications

核磁気共鳴 (NMR) 分光法

Source: Laboratory of Dr. Henrik Sund&#233;n &#8211; Chalmers University of Technology

Nuclear magnetic resonance (NMR) spectroscopy is a vital analysis ...

Methods to Identify the NMR Resonances of the ¹³C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

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