Reaction Of Amino Acids Research Articles

Peracetic acid disinfection induces antibiotic-resistant E. coli into VBNC state but ineffectively eliminates the transmission potential of ARGs

The occurrence of a viable but nonculturable (VBNC) state in antibiotic-resistant E. coli (AR E. coli) and inefficient degradation of their antibiotic resistance genes (ARGs) may cause potential health risks during disinfection. Peracetic acid (PAA) is an alternative disinfectant for replacing chlorine-based oxidants in wastewater treatment, and the potential of PAA to induce a VBNC state in AR E. coli and to remove the transformation functionality of ARGs were investigated for the first time. Results show that PAA exhibits excellent performance in inactivating AR E. coli (over 7.0-logs) and persistently inhibiting its regeneration. After PAA disinfection, insignificant changes in the ratio of living to dead cells (∼4%) and the level of cell metabolism, indicating that AR E. coli were induced into VBNC states. Unexpectedly, PAA was found to induce AR E. coli into VBNC state by destroying the proteins containing reactive amino acids at thiol, thioether and imidazole groups, rather than the result of membrane damage, oxidative stress, lipid destruction and DNA disruption in the conventional disinfection processes. Moreover, the result of poor reactivity between PAA and plasmid strands and bases confirmed that PAA hardly reduced the abundance of ARGs and damaged the plasmid's integrity. Transformation assays and real environment validation indicated that PAA-treated AR E. coli could release large abundance of naked ARGs with high-efficiency transformation functionality (∼5.4 × 10–4 - ∼8.3 × 10–6) into the environment. This study has significant environmental implications for assessing the transmission of antimicrobial resistance during PAA disinfection.

Genetic code expansion in E. coli enables production of a functional ‘ready-to-click’ T cell receptor-specific scFv

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable VH and VL domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid Nε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition (‘click chemistry’). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a ‘ready-to-click’ scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

Bioorthogonal Tethering Enhances Drug Fragment Affinity for G Protein-Coupled Receptors in Live Cells.

G protein-coupled receptors (GPCRs) modulate diverse cellular signaling pathways and are important drug targets. Despite the availability of high-resolution structures, the discovery of allosteric modulators remains challenging due to the dynamic nature of GPCRs in native membranes. We developed a strategy to covalently tether drug fragments adjacent to allosteric sites in GPCRs to enhance their potency and enable fragment-based drug screening in cell-based systems. We employed genetic code expansion to site-specifically introduce noncanonical amino acids with reactive groups in C-C chemokine receptor 5 (CCR5) near an allosteric binding site for the drug maraviroc. We then used molecular dynamics simulations to design heterobifunctional maraviroc analogues consisting of a drug fragment connected by a flexible linker to a reactive moiety capable of undergoing a bioorthogonal coupling reaction. We synthesized a library of these analogues and employed the bioorthogonal inverse electron demand Diels-Alder reaction to couple the analogues to the engineered CCR5 in live cells, which were then assayed using cell-based signaling assays. Tetherable low-affinity maraviroc fragments displayed an increase in potency for CCR5 engineered with reactive unnatural amino acids that were adjacent to the maraviroc binding site. The strategy we describe to tether novel drug fragments to GPCRs should prove useful to probe allosteric or cryptic binding site functionality in fragment-based GPCR-targeted drug discovery.

ROS-Triggered Gel-Sol Transition and Kinetics-Controlled Cargo Release by Methionine-Containing Peptides.

The gel-sol transition of self-assembling peptides is a useful switch for environment-dependent drug release. For their applications, kinetics control of the responses is important for matching the velocity of release to the target biological events. Here we demonstrate the chemical control of redox-triggered gel-sol transition kinetics of self-assembling peptides by altering the amino acid sequence. Amphiphilic peptides were developed in which a methionine residue was located in the middle (JigSAP-IMI) or near the N terminus (JigSAP-MII). Both peptides formed hydrogels under physiological conditions-forming β-sheet-based supramolecular nanofibers. In contrast, the oxidized forms remained in the solution state under identical conditions-adopting α-helix-rich secondary structures. Upon oxidation with H2 O2 , a reactive oxygen species, JigSAP-MII showed a faster gel-to-sol transition and cargo-releasing than JigSAP-IMI, thus indicating that the phase-transition and releasing kinetics of self-assembling peptides can be rationally controlled by the position of the reactive amino acid residue.

Dissecting metabotropic glutamate receptor activation by single molecule FRET using minimally invasive labeling strategies.

Metabotropic glutamate receptors (mGluR) regulate neuronal excitability and synaptic transmission by sensing L-glutamate - the major excitatory neurotransmitter in the central nervous system. Their crucial role for synaptic function makes them attractive targets for the treatment of numerous neurological and psychiatric diseases including for instance anxiety, depression, schizophrenia and addiction. To explore mGluR activation, we monitored its conformational changes using single molecule two and three color FRET with submillisecond resolution. We established multiple, minimally invasive conformational FRET sensors through bioorthogonal double labeling of two reactive and different non-canonical amino acids after incorporation by genetic code expansion. These sensors report on the initial step of mGluR2 activation, including the reorientation of the upper and the lower lobes of the venus flytrap domain (VFT) in an intersubunit fashion and its closure in an intrasubunit fashion. By studying ligand induced conformational changes on single receptors, we show that the natural full agonist glutamate is sufficient to close the VFTs but a synergy with synthetic positive allosteric modulators or the G protein, is required for full activation. Our study highlights the power of minimally invasive, ncAA-based, bioorthogonal labeling to dissect domain-specific conformational rearrangements of single GPCRs using smFRET.

Selective thiazoline peptide cyclisation compatible with mRNA display and efficient synthesis.

Peptide display technologies are a powerful method for discovery of new bioactive sequences, but linear sequences are often very unstable in a biological setting. Macrocyclisation of such peptides is beneficial for target affinity, selectivity, stability, and cell permeability. However, macrocyclisation of a linear hit is unreliable and requires extensive structural knowledge. Genetically encoding macrocyclisation during the discovery process is a better approach, and so there is a need for diverse cyclisation options that can be deployed in the context of peptide display techniques such as mRNA display. In this work we show that meta-cyanopyridylalanine (mCNP) can be ribosomally incorporated into peptides, forming a macrocycle in a spontaneous and selective reaction with an N-terminal cysteine generated from bypassing the initiation codon in translation. This reactive amino acid can also be easily incorporated into peptides during standard Fmoc solid phase peptide synthesis, which can otherwise be a bottleneck in transferring from peptide discovery to peptide testing and application. We demonstrate the potential of this new method by discovery of macrocyclic peptides targeting influenza haemagglutinin, and molecular dynamics simulation indicates the mCNP cross-link stabilises a beta sheet structure in a representative of the most abundant cluster of active hits. Cyclisation by mCNP is also shown to be compatible with thioether macrocyclisation at a second cysteine to form bicycles of different architectures, provided that cysteine placement reinforces selectivity, with this bicyclisation happening spontaneously and in a controlled manner during peptide translation. Our new approach generates macrocycles with a more rigid cross-link and with better control of regiochemistry when additional cysteines are present, opening these up for further exploitation in chemical modification of in vitro translated peptides, and so is a valuable addition to the peptide discovery toolbox.

Harnessing selenocysteine to enhance microbial cell factories for hydrogen production.

Hydrogen is a clean, renewable energy source, that when combined with oxygen, produces heat and electricity with only water vapor as a biproduct. Furthermore, it has the highest energy content by weight of all known fuels. As a result, various strategies have engineered methods to produce hydrogen efficiently and in quantities that are of interest to the economy. To approach the notion of producing hydrogen from a biological perspective, we take our attention to hydrogenases which are naturally produced in microbes. These organisms have the machinery to produce hydrogen, which when cleverly engineered, could be useful in cell factories resulting in large production of hydrogen. Not all hydrogenases are efficient at hydrogen production, and those that are, tend to be oxygen sensitive. Therefore, we provide a new perspective on introducing selenocysteine, a highly reactive proteinogenic amino acid, as a strategy towards engineering hydrogenases with enhanced hydrogen production, or increased oxygen tolerance.

Bioactive Nonthermal Biocompatible Plasma Enhances Migration on Human Gingival Fibroblasts.

This study hypothesizes that the application of low-dose nonthermal biocompatible dielectric barrier discharge plasma (DBD-NBP) to human gingival fibroblasts (HGFs) will inhibit colony formation but not cell death and induce matrix metalloproteinase (MMP) expression, extracellular matrix (ECM) degradation, and subsequent cell migration, which can result in enhanced wound healing. HGFs treated with plasma for 3min migrate to each other across the gap faster than those in the control and 5-min treatment groups on days 1 and 3. The plasma-treated HGFs show significantly high expression levels of the cell cycle arrest-related p21 gene and enhanced MMP activity. Focal adhesion kinase (FAK) mediated attenuation of wound healing or actin cytoskeleton rearrangement, and plasma-mediated reversal of this attenuation support the migratory effect of DBD-NBP. Further, this work performs computer simulations to investigate the effect of oxidation on the stability and conformation of the catalytic kinase domain (KD) of FAK. It is found that the oxidation of highly reactive amino acids (AAs) Cys427, Met442, Cys559, Met571, Met617, and Met643 changes the conformation and increases the structural flexibility of the FAK protein and thus modulates its function and activity. Low-dose DBD-NBP-induces host cell cycle arrest, ECM breakdown, and subsequent migration, thus contributing to the enhanced wound healing process.

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.

Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis-up to 93%-of Gb3+ tumor cells in vitro. This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

Near-Infrared Fluorescence Probe with a New Recognition Moiety for Specific Detection and Imaging of Aldehyde Dehydrogenase Expecting the Identification and Isolation of Cancer Stem Cells.

Aldehyde dehydrogenase (ALDH) is a vital enzyme that converts aldehyde to acetic acid during alcohol metabolism. ALDH is also a cellular marker of cancer stem cells (CSCs), which plays an important role in cancer diagnosis and prognosis assessment. Therefore, there is a need to explore convenient, selective, and sensitive methods for the detection and imaging of ALDH. Because of the low background fluorescence and high penetration, near-infrared (NIR) fluorescent probes are powerful tools for the detection of ALDH. Until now, only one NIR fluorescent probe has been reported for detecting ALDH. Hence, we synthesized a novel NIR fluorescent probe, Probe-ALDH, by linking the new specific recognition moiety 4-hydroxymethyl benzaldehyde with NIR fluorophore AXPI. Compared with the existing ALDH fluorescent probes, Probe-ALDH has excellent properties, such as a new specific recognition moiety without the substitution of benzaldehyde, a simple synthesis method, emission wavelength in the NIR region, reaction time of only 30 min, and a detection limit as low as 0.03 U·mL-1, which is better than those of the previously reported probes. The probe effectively eliminates the interference from reactive oxygen species (ROS), amino acids, and amines. More importantly, the flow cytometry results showed that Probe-ALDH has great potential applications in the identification and isolation of CSCs. Ultimately, it was successfully applied to the imaging analysis of endogenous ALDH in HepG2 cells by the addition of inhibitor disulfiram. The excellent performance of Probe-ALDH makes it a promising candidate for drug discovery, cancer diagnosis, and so forth.

Multi-walled carbon nanotubes as a platform for Immunoglobulin G attachment

Nanomaterials have been extensively used in different applications due to their peculiar characteristics and nanoscale dimensions. Among nanoparticles, carbon-based nanomaterials are becoming highly attractive for biomedical applications such as diagnosis, tissue engineering, drug delivery, and biosensing. The conjugation of carbon-based nanomaterials with antibodies combines the properties of these materials with the specific and selective recognition ability of the antibodies to antigens. The present work proposes a process intensification approach for immunoglobulin G (IgG present in rabbit serum) attachment on multi-walled carbon nanotubes (MWCNTs) in a single step. The effect of several parameters, namely MWCNTs external diameter, rabbit serum concentration, MWCNTs functionalization and pH value, on the IgG attachment yield was evaluated. The dilution of rabbit serum decreased other protein attachment, namely rabbit serum albumin (RSA), while increasing the IgG yield to 100%. The interaction mechanisms between IgG and MWCNTs were evaluated at pH 5.0 to 8.0. The protonation of IgG amino acids indicates that N-term are the most reactive amino acids in the antibody structure. The identification of the N-term reactivity at pH 8.0 allows to indicate a possible orientation of the antibody over the MWCNTs surface, described as “end-on”. Since the amount of RSA attached to MWNT decreased with the increase in serum dilution, the IgG orientation and amine activity was not affected. This orientation demonstrates that the IgG attachment over the surface of the MWCNTs could be an effective strategy to maintain the antigen recognition by the antibody, and to be used for biomedical applications.

Neat Protein Single-Chain Nanoparticles from Partially Denatured BSA.

The main challenge for the preparation of protein single-chain nanoparticles (SCNPs) is the natural complexity of these macromolecules. Herein, we report the suitable conditions to produce "neat" bovine serum albumin (BSA) single-chain nanoparticles (SCNPs) from partially denatured BSA, which involves denaturation in urea and intramolecular cross-linking below the overlap concentration. We use two disuccinimide ester linkers containing three and six methylene spacer groups: disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS), respectively. Remarkably, the degree of internal cross-linking can be followed simply and efficiently via 1H NMR spectroscopy. The associated structural changes-as probed by small-angle neutron scattering (SANS)-reveal that the denatured protein has a random-like coil conformation, which progressively shrinks with the addition of DSG or DSS, thus allowing for size control of the BSA-SCNPs with radii of gyration down to 5.4 nm. The longer cross-linker exhibits slightly more efficiency in chain compaction with a somewhat stronger size reduction but similar reactivity at a given cross-linker concentration. This reliable method is applicable to a wide range of compact proteins since most proteins have appropriate reactive amino acids and denature in urea. Critically, this work paves the way to the synthesis of "neat", biodegradable protein SCNPs for a range of applications including nanomedicine.

Synthesis of L-cyclic tetrapeptides by backbone amide activation CyClick strategy

Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.

Ferroptosis Inducers for Prostate Cancer Therapy.

Lipid peroxidation-driven iron-dependent ferroptosis is a regulated cell death mechanism implicated in numerous diseases, such as neurological diseases, kidney injury, ischemia, and tumors, including prostate cancer. The cellular mechanisms of ferroptosis are strongly associated with iron, reactive oxygen species and amino acid metabolic pathways. Several compounds, namely ferroptosis inducers, impact these pathways and trigger ferroptosis by i) inhibiting Xc - transporter system, ii) impairing GPX4 functions and iii) oxidizing iron and polyunsaturated phospholipids. Preclinical studies show that in combination with conventional anticancer drugs, ferroptosis inducers are effective in prostate cancer and in combating the progression towards the castration-resistant disease. This review overviews the mechanisms implicated in ferroptosis and discusses the findings achieved in prostate cancer.

Amino acid functionalized benzanthrone dyes: Synthesis and photophysical study

The substitution reaction of 3-N-(2-chloroacetamido)benzanthrone with a number of the amino acids is reported. The possibility of using this halogenated benzanthrone for fluorescent labelling of amino acids has been studied. Arginine is the most reactive amino acid in the reaction, as it was found in the study. The spectral behavior of new benzanthrone derivatives depends on the polarity of the medium, exhibiting fluorescent solvatochromism due to intramolecular charge transfer from the substituted amino group to the carbonyl group. The results obtained indicate that the synthesized compounds are promising for studying peptides and proteins, as well as for visualizing biological objects.

Escherichia coli Uses a Dedicated Importer and Desulfidase To Ferment Cysteine.

ABSTRACTCyuA of Escherichia coli is an inducible desulfidase that degrades cysteine to pyruvate, ammonium, and hydrogen sulfide. Workers have conjectured that its role may be to defend bacteria against the toxic effects of cysteine. However, cyuA sits in an operon alongside cyuP, which encodes a cysteine importer that seems ill suited to protecting the cell from environmental cysteine. In this study, transport measurements established that CyuP is a cysteine-specific, high-flux importer. The concerted action of CyuP and CyuA allowed anaerobic E. coli to employ cysteine as either the sole nitrogen or the sole carbon/energy source. CyuA was essential for this function, and although other transporters can slowly bring cysteine into the cell, CyuP-proficient cells outcompeted cyuP mutants. Cells immediately consumed the ammonia and pyruvate that CyuA generated, with little or none escaping from the cell. The expression of the cyuPA operon depended upon both CyuR, a cysteine-activated transcriptional activator, and Crp. This control is consistent with its catabolic function. In fact, the cyuPA operon sits immediately downstream of the thrABCDEFG operon, which allows the analogous fermentation of serine and threonine; this arrangement suggests that this gene cluster may have moved jointly through the anaerobic biota, providing E. coli with the ability to ferment a limited set of amino acids. Interestingly, both the cyu- and thr-encoded pathways depend upon oxygen-sensitive enzymes and cannot contribute to amino acid catabolism in oxic environments.

THE USE OF BIO-MOLECULAR SUBSTANCE OF MARINE BIOTA AS AN ALTERNATIVE EARLY INDICATOR OF OIL POLLUTED ENVIRONMENT: A NEW APPROACH FOR MONITORING CONSIDERATION

THE USE OF BIO-MOLECULAR SUBSTANCE OF MARINE BIOTA AS AN ALTERNATIVE EARLY INDICATOR OF OIL POLLUTED ENVIRONMENT: A NEW APPROACH FOR MONITORING CONSIDERATION

Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization

蛋白质主要以复合物的形式参与各项生命活动。化学交联质谱(CXMS)技术作为近年来新兴的蛋白质复合物解析技术,不仅可实现蛋白质复合物规模化解析,而且普遍适用于任意相对分子质量和纯度的蛋白质复合物样品,因此已成为X-射线晶体衍射技术、冷冻电镜技术等蛋白质复合物解析经典技术的重要补充。目前,CXMS主要采用胰蛋白酶将交联后的蛋白质复合物进行酶切,并进行质谱鉴定。然而鉴定到的交联肽段谱图中b/y特征碎片离子的鉴定数目不足,且因肽段序列匹配连续性较差导致谱图可信度相对较低,影响了蛋白质复合物交联位点的鉴定精准度。该文基于镜像酶正交切割的互补特性,采用胰蛋白酶镜像酶与胰蛋白酶联合酶切的方法,对交联后的蛋白质复合物分别进行酶切,再利用液相色谱-质谱联用技术对酶解产生的交联肽段进行鉴定,进而实现蛋白质复合物的组成、相互作用和结构位点距离约束的解析。对牛血清白蛋白和大肠杆菌全蛋白的交联肽段的分析结果表明,该方法通过增加交联肽段谱图中特征碎裂离子的数目和连续性,鉴定到的大肠杆菌全蛋白的交联位点,较单一胰蛋白酶酶切,提高了16%。因此,镜像酶正交酶切策略能有效提高交联肽段的鉴定准确度和覆盖度,有望为实现规模化的蛋白质复合物精准解析提供新思路。

Drop-off-reinitiation triggered by EF-G-driven mistranslocation and its alleviation by EF-P.

In ribosomal translation, peptidyl transfer occurs between P-site peptidyl-tRNA and A-site aminoacyl-tRNA, followed by translocation of the resulting P-site deacylated-tRNA and A-site peptidyl-tRNA to E and P site, respectively, mediated by EF-G. Here, we report that mistranslocation of P-site peptidyl-tRNA and A-site aminoacyl-tRNA toward E and A site occurs when high concentration of EF-G triggers the migration of two tRNAs prior to completion of peptidyl transfer. Consecutive incorporation of less reactive amino acids, such as Pro and d-Ala, makes peptidyl transfer inefficient and thus induces the mistranslocation event. Consequently, the E-site peptidyl-tRNA drops off from ribosome to give a truncated peptide lacking the C-terminal region. The P-site aminoacyl-tRNA allows for reinitiation of translation upon accommodation of a new aminoacyl-tRNA at A site, leading to synthesis of a truncated peptide lacking the N-terminal region, which we call the ‘reinitiated peptide’. We also revealed that such a drop-off-reinitiation event can be alleviated by EF-P that promotes peptidyl transfer of Pro. Moreover, this event takes place both in vitro and in cell, showing that reinitiated peptides during protein synthesis could be accumulated in this pathway in cells.

Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea.

The study of protein production and degradation in a quantitative and time-dependent manner is a major challenge to better understand cellular physiological response. Among available technologies bioorthogonal noncanonical amino acid tagging (BONCAT) is an efficient approach allowing for time-dependent labeling of proteins through the incorporation of chemically reactive noncanonical amino acids like L-azidohomoalanine (L-AHA). The azide-containing amino-acid derivative enables a highly efficient and specific reaction termed click chemistry, whereby the azide group of the L-AHA reacts with a reactive alkyne derivate, like dibenzocyclooctyne (DBCO) derivatives, using strain-promoted alkyne-azide cycloaddition (SPAAC). Moreover, available DBCO containing reagents are versatile and can be coupled to fluorophore (e.g., Cy7) or affinity tag (e.g., biotin) derivatives, for easy visualization and affinity purification, respectively.Here, we describe a step-by-step BONCAT protocol optimized for the model archaeon Haloferax volcanii , but which is also suitable to harness other biological systems. Finally, we also describe examples of downstream visualization, affinity purification of L-AHA-labeled proteins and differential expression analysis.In conclusion, the following BONCAT protocol expands the available toolkit to explore proteostasis using time-resolved semiquantitative proteomic analysis in archaea .