Labeling Protocol Research Articles

Chemoproteomic Profiling of Adenylation Domain Functions in Gramicidin S-Producing Non-ribosomal Peptide Synthetases.

Many amino acid-containing natural products are biosynthesized by large, multifunctional enzymes known as non-ribosomal peptide synthetases (NRPSs). Adenylation (A) domains in NRPSs are responsible for the incorporation of amino acid building blocks and can be considered as engineering domains; therefore, advanced techniques are required to not only rapidly verify expression and folding, but also accelerate the functional prediction of the A-domains in lysates from native and heterologous systems. We recently developed activity-based protein profiling (ABPP) of NRPSs that offers a simple and robust analytical platform for A-domains and provides insights into their enzyme-substrate specificity. In this chapter, we describe the design and synthesis of these ABPP probes and provide a summary of our work on the development of a series of protocols for labeling, visualizing, and analyzing endogenous NRPSs in complex biological systems.

The linkage-type and the exchange molecule affect the protein-labeling efficiency of iminoboronate probes.

Reversible bioorthogonal conjugation reactions have been exploited in the chemoproteomic field to prepare protein labeling reagents and to visualize labeled proteins. We recently demonstrated that reversible iminoboronates can be used to prepare probes from fragment libraries and that the linkage subsequently can be used to detect the labeled proteins. In this study, we determined the effect of the stability of the iminoboronate linkage on the efficiency of the labeling protocol. Our study reveals that the linkage should be stable enough to allow for efficient targeting, but should be labile enough to detect the labeled protein. Acyl hydrazides were identified as the most suitable handles for the probe synthesis step. Anthranilic hydrazides and N-hydroxy semicarbazides were found to be the most efficient read-out molecules. With these novel exchange molecules, native probe-labeled proteins could be visualized under physiological conditions.

Preparing recombinant "Split AEP" for protein labeling.

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity. Here, we report a novel preparation protocol, in which the cap and catalytically active core domains are produced as separate entities. The active enzyme can be obtained in two chromatographic steps, immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC), with no acid activation required, thereby shortening the purification procedure from at least 2 days to less than 6h. In addition to the original C247A mutation which enhanced reaction with various amino nucleophiles, an extra D29E mutation was introduced to prevent self-cleavage, which led to noticeable improvements in homogeneity and activity of the enzyme. Indeed, the resulting "split AEP" (i.e., core domain of OaAEP1-D29E/C247A) exhibited improved catalytic efficiency constant (kcat/KM) that was found to be ∼3-fold higher than that of the original acid-activated counterpart (OaAEP1-C247A). Furthermore, we described a protein labeling protocol that couples the enzymatic reaction with an irreversible chemical transformation, thereby enabling high conversion of labeled protein with a lowered amount of reagent. Precisely, an alternative Asn-Cys-Leu (NCL) recognition sequence was used for substrate recognition. As the byproduct contains an N-terminal cysteine, it can be transformed into an inert 1,2 aminothiol motif by reacting with formylphenyl boronic acid (FPBA). Finally, the opportunities and challenges associated with the use of asparaginyl ligase are discussed.

Synthesis, Optical Properties, and Antiproliferative Evaluation of NBD-Triterpene Fluorescent Probes.

A fluorescent labeling protocol for hydroxylated natural compounds with promising antitumor properties has been used to synthesize, in yields of 72-86%, 12 derivatives having fluorescent properties and biological activity. The reagent used for the synthesis of these fluorescent derivatives was 7-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl). The linkers employed to bind the NBD-Cl reagent to the natural compounds were ω-amino acids (Aa) of different chain lengths. The natural triterpene compounds chosen were oleanolic and maslinic acid, as their corresponding 28-benzylated derivatives. Thus, 12 NBD-Aa-triterpene conjugates have been studied for their optical fluorescence properties and their biological activities against cell proliferation in three cancer cell lines (B16-F10, HT-29, and HepG2), compared with three nontumor cell lines (HPF, IEC-18, and WRL68) from different tissues. The results of the fluorescence study have shown that the best fluorescent labels are those in which the ω-amino acid chain is shorter, and the carboxylic group is not benzylated. Analysis by confocal microscopy showed that these compounds were rapidly incorporated into cells in all three cancer cell lines, with these same derivatives showing the highest toxicity against the cancer cell lines tested. Then, the fluorescent labeling of these NBD-Aa-triterpene conjugates enabled their uptake and subcellular distribution to be followed in order to probe in detail their biological properties at the cellular and molecular level.

Reliability of BBB water exchange rate assessed by diffusion‐prepared pseudo‐continuous ASL (DP‐pCASL): Preliminary results from the Cognitive Recovery After Elective Surgery (CREATES) Study

AbstractBackgroundImpaired blood‐brain barrier (BBB) function is hypothesized to be an important mechanism in the development of perioperative neurocognitive disorders (postoperative delirium and postoperative cognitive impairment), as well as in the onset and progression of Alzheimer’s (AD) and cerebrovascular disease. Non‐contrast neuroimaging methods have shown promise for detecting subtle alterations in BBB permeability; however, test‐retest reliability has not been well‐established. Here we report preliminary results for BBB water exchange rate (kw ) values from a novel diffusion‐prepared 3D pseudo‐continuous arterial spin labeling (DP‐pCASL) protocol with two different post‐labeling delays (PLD), and compared test‐retest reliability results with the original approach with a single PLD.MethodThe DP‐pCASL pulse sequence (resolution=3.5x3.5x8mm3, 10 slices) has been previously described (Shao X, et al., 2019). kw was calculated from the ratio of intra‐ and extra‐vascular perfusion signals using the original protocol with 1‐PLD [1.8 sec]; and the new protocol with 2‐PLDs [1.8 and 2.1 sec]. Total scan time was 15 minutes.Participants were healthy older adult controls enrolled in Cognitive Recovery After Elective Surgery (CREATES) study, a longitudinal study of relationships between BBB dysfunction and perioperative neurocognitive disorders following major elective surgery. Seventeen participants (11 females, age=78.5±5.4 yrs) underwent test‐retest MRI scans (∼30 days apart) on a Siemens 3T Prisma Fit system (Erlangen, Germany). kw maps were normalized into the MNI space, and regional analysis was performed for cortical gray/white matter, frontal/temporal/parietal areas, and nine subcortical brain regions. Test‐retest reliability was evaluated by intra‐class correlation coefficients (ICC).ResultWhole brain and regional kw values were calculated for the two protocols (Table 1). Test‐retest reliability increased using the 2‐PLD protocol. Whole brain kw ICC was 0.7 (good reliability; reference range, 0.5‐0.74) vs. 0.87 (excellent reliability; reference range, ≥ 0.75) for 1‐PLD and 2‐PLD protocols, respectively (Figure 1). ICC values increased across all brain regions with the 2‐PLD protocol (Figure 2).ConclusionIn a modest sample of cognitively healthy older adults, we found that measurement reliability of whole brain and regional kw values was notably improved by using the 2‐PLD DP‐pCASL protocol. These preliminary findings warrant confirmation in larger cohorts with and without late‐life cognitive disorders.

EVAnalyzer: High content imaging for rigorous characterisation of single extracellular vesicles using standard laboratory equipment and a new open-source ImageJ/Fiji plugin.

Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

Proximity-Dependent In Vivo Biotin Labeling for Interactome Mapping in Marchantia polymorpha.

Weak or transient protein-protein interactions (PPIs) are involved in a manifold of cellular processes in all living organisms, including plants. However, many of these interactions may remain undiscovered by co-immunoprecipitation (Co-IP) approaches due to their low binding affinities or transitory nature. Enzyme-mediated proximity-dependent in vivo biotin labeling can be a powerful strategy to efficiently capture weak and transient PPIs and has been successfully applied in different model angiosperm species. Here, we provide an optimized and robust protocol for biotin ligase-mediated proximity labeling for interactome mapping in the model liverwort Marchantia polymorpha.

Cesium Amide-Catalyzed Selective Deuteration of Benzylic C-H Bonds with D2 and Application for Tritiation of Pharmaceuticals.

Hydrogen isotope exchange (HIE) represents one of the most attractive labeling methods to synthesize deuterium- and tritium-labeled compounds. Catalytic HIE methods that enable site-selective C-H bond activation and exchange labeling with gaseous isotopes D2 and T2 are of vital importance, in particular for high-specific-activity tritiation of pharmaceuticals. As part of our interest in exploring s-block metals for catalytic transformations, we found CsN(SiMe3 )2 to be an efficient catalyst for selective HIE of benzylic C-H bonds with D2 gas. The reaction proceeds through a kinetic deprotonative equilibrium that establishes an exchange pathway between C-H bonds and D2 gas. By virtue of multiple C-H bonds activation and high activity (isotope enrichment up to 99 %), the simple cesium amide catalyst provided a very powerful and practically convenient labeling protocol for synthesis of highly deuterated compounds and high-specific-activity tritiation of pharmaceuticals.

Cesium Amide‐Catalyzed Selective Deuteration of Benzylic C‐H Bonds with D2 and Application for Tritiation of Pharmaceuticals

AbstractHydrogen isotope exchange (HIE) represents one of the most attractive labeling methods to synthesize deuterium‐ and tritium‐labeled compounds. Catalytic HIE methods that enable site‐selective C−H bond activation and exchange labeling with gaseous isotopes D2 and T2 are of vital importance, in particular for high‐specific‐activity tritiation of pharmaceuticals. As part of our interest in exploring s‐block metals for catalytic transformations, we found CsN(SiMe3)2 to be an efficient catalyst for selective HIE of benzylic C−H bonds with D2 gas. The reaction proceeds through a kinetic deprotonative equilibrium that establishes an exchange pathway between C−H bonds and D2 gas. By virtue of multiple C−H bonds activation and high activity (isotope enrichment up to 99 %), the simple cesium amide catalyst provided a very powerful and practically convenient labeling protocol for synthesis of highly deuterated compounds and high‐specific‐activity tritiation of pharmaceuticals.

DIGE Analysis of Clinical Specimens.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2D-GE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The use of an internal pooled standard makes 2D-DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. Technical limitations of this technique (i.e., underrating of low abundant, high molecular mass and integral membrane proteins) are counterbalanced by the incomparable separation power which allows proteoforms and unknown PTM (posttranslational modification) identification. Moreover, the image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

SILAC-Based Quantitative Proteomic Analysis of Drosophila Embryos.

The fruit fly Drosophila melanogaster represents a classic genetic model organism that is amenable to a plethora of comprehensive analyses including proteomics. SILAC-based quantitative proteomics is a powerful method to investigate the translational and posttranslational regulation ongoing in cells, tissues, organs, and whole organisms. Here we describe a protocol for routine SILAC labeling of Drosophila adults within one generation to produce embryos with a labeling efficiency of over 92%. In combination with genetic selection markers, this method permits the quantification of translational and posttranslational changes in embryos mutant for developmental and disease-related genes.

Using Activity-Based Proteomics for the Quantification of Deubiquitinases in Animal Tissue.

Ubiquitination isapost-translational modification, thatregulates essential cellular functions, and the enzymes that control the removal of this modification, deubiquitinases (DUBs), have been well described for the model organisms. However, the information about DUBs is still largely lacking for the non-model organisms, such as agriculturally relevant animals. To understand the expression of these enzymes in animal tissues, we have used chemical proteomics which can be used to identify biologically active DUBs present in tissues based on their reactivity with the activity-based probes (ABPs). Here we describe a sample preparation protocol for ABP-based purification of DUBs from animal tissue using two approaches to homogenize and lyse the animal tissue compatible with ABP labeling of DUBs, including anultrasonication-based tissue processing method and bead-beating method. Both of these methods retain the enzymatic activity of DUBs. In addition, we describe a protocol for ABP labeling of DUBs in tissue lysates and the immunoprecipitation of the probe-reactive DUBs that can be used along with mass spectrometric identification of proteins and the detection of these DUBs by Western blotting.

Evaluation of Tetrofosmin Cold Kit Fractionation Using Alternative New Method of Quality Control for Testing Radiochemical Purity

Objective The aim of this study is to establish a method for the fractionation of tetrofosmin cold kit under different storage conditions and to optimize an alternate chromatography method from the reference method to test radiochemical purity (RCP). Materials and Methods Tetrofosmin cold kit vial was fractionated aseptically in six equal fractions and stored in vials and syringes. To test the stability of the reconstituted solution for a longer duration, the mother vials and syringes were stored at two different temperatures, that is, at 4°C and at -20°C till further used. Radiolabeling of fractionated tetrofosmin was performed as per the standard labeling protocol. Radionuclide purity, radioassay, and pH were tested. Radiolabeling efficiency and RCP were determined by paper chromatography. Results Radionuclide purity of eluate was greater than 99.9%. The pH of technetium-99m (Tc-99m) eluate and Tc-99m tetrofosmin was between 4.5-7.5 and 7.5-9.5, respectively. The deviation in the radioactivity during all measurements was less than 1%. The kits fractioned in glass vials resulted in higher radiolabeling yield and RCP as compared with kits fractionated in syringes. The RCP of glass vial versus syringe was observed to be greater than 95 versus 90% and 95 versus 80% at -20°C and 4°C, respectively. Conclusion Tetrofosmin kit can be used in a cost-effective manner by fractionation. One tetrofosmin vial can be used in six fractions for up to 15 days when stored at -20°C and 4°C freezer temperature. The alternative method to check the RCP of Tc-99m tetrofosmin is safer and less time consuming as compared with the reference method.

Blind spots on western blots: Assessment of common problems in western blot figures and methods reporting with recommendations to improve them.

Western blotting is a standard laboratory method used to detect proteins and assess their expression levels. Unfortunately, poor western blot image display practices and a lack of detailed methods reporting can limit a reader's ability to evaluate or reproduce western blot results. While several groups have studied the prevalence of image manipulation or provided recommendations for improving western blotting, data on the prevalence of common publication practices are scarce. We systematically examined 551 articles published in the top 25% of journals in neurosciences (n = 151) and cell biology (n = 400) that contained western blot images, focusing on practices that may omit important information. Our data show that most published western blots are cropped and blot source data are not made available to readers in the supplement. Publishing blots with visible molecular weight markers is rare, and many blots additionally lack molecular weight labels. Western blot methods sections often lack information on the amount of protein loaded on the gel, blocking steps, and antibody labeling protocol. Important antibody identifiers like company or supplier, catalog number, or RRID were omitted frequently for primary antibodies and regularly for secondary antibodies. We present detailed descriptions and visual examples to help scientists, peer reviewers, and editors to publish more informative western blot figures and methods. Additional resources include a toolbox to help scientists produce more reproducible western blot data, teaching slides in English and Spanish, and an antibody reporting template.

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

New insights on energetic properties of graphene oxide (GO) materials and their safety and environmental risks

Graphene oxide (GO) has been recognized as a thermally unstable and energetic material, but surprisingly its environmental and safety risks were not fully explored, defined, and regulated. In this study, systematic explosivity and flammability characterizations of commercial GO materials were conducted to evaluate the influence of key parameters such as physical forms (paste, powders, films, and aerogels), temperature, heating rate, mass, and heating environment, as well as their potential safety and environmental impacts. Results based on thermogravimetric analysis (TGA) showed that GO in paste and powder forms have lower temperature thresholds (>180–192 °C) to initiate micro-explosions compared to GO film and aerogels (> 205 °C and 213 °C) regardless of the environment (inert, air, or oxygen). The observed explosive behavior can be explained by thermal runaway reactions as a result of thermal deoxygenation and decomposition of oxygen functional groups. Flammability rating and limiting oxygen index (LOI) results confirmed that GO films are flammable materials that can spontaneously propagate flame in a low oxygen environment (~11 %). These results provided new insights about potential safety and environmental risks of GO materials, which somehow were not considered, suggesting urgent actions to improve current safety protocols for labeling, handling, transporting, and storage practices from manufacturers to the end-users.

TUBright: A Peptide Probe for Imaging Microtubules.

In vitro assays using reconstituted microtubules have provided molecular insights into the principles of microtubule dynamics and the roles of microtubule-associated proteins. Emerging questions that further uncover the complexity in microtubule dynamics, especially those on tubulin isotypes and post-translational modifications, raise new technical challenges on how to visualize microtubules composed of tubulin purified from limited sources, primarily due to the low efficiency of the conventional tubulin labeling protocol. Here, we develop a peptide probe, termed TUBright, that labels in vitro reconstituted microtubules. TUBright, when coupled with different fluorescent dyes, provides flexible labeling of microtubules with a high signal-to-noise ratio. TUBright does not interfere with the dynamic behaviors of microtubules and microtubule-associated proteins. Therefore, TUBright is a useful tool for imaging microtubules, making it feasible to use tubulin from limited sources for answering many open questions on microtubule dynamics.

Automatic segmentation of white matter hyperintensities in routine clinical brain MRI by 2D VB-Net: A large-scale study.

White matter hyperintensities (WMH) are imaging manifestations frequently observed in various neurological disorders, yet the clinical application of WMH quantification is limited. In this study, we designed a series of dedicated WMH labeling protocols and proposed a convolutional neural network named 2D VB-Net for the segmentation of WMH and other coexisting intracranial lesions based on a large dataset of 1,045 subjects across various demographics and multiple scanners using 2D thick-slice protocols that are more commonly applied in clinical practice. Using our labeling pipeline, the Dice consistency of the WMH regions manually depicted by two observers was 0.878, which formed a solid basis for the development and evaluation of the automatic segmentation system. The proposed algorithm outperformed other state-of-the-art methods (uResNet, 3D V-Net and Visual Geometry Group network) in the segmentation of WMH and other coexisting intracranial lesions and was well validated on datasets with thick-slice magnetic resonance (MR) images and the 2017 medical image computing and computer assisted intervention WMH Segmentation Challenge dataset (with thin-slice MR images), all showing excellent effectiveness. Furthermore, our method can subclassify WMH to display the WMH distributions and is very lightweight. Additionally, in terms of correlation to visual rating scores, our algorithm showed excellent consistency with the manual delineations and was overall better than those from other competing methods. In conclusion, we developed an automatic WMH quantification framework for multiple application scenarios, exhibiting a promising future in clinical practice.

In capillary labeling and online electrophoretic separation of N-glycans from glycoproteins.

In this study, we present a new approach for in-capillary fluorescent labeling of N-glycans prior to their analysis with CE coupled with laser-induced fluorescent detection. This integrated approach allows using a CE capillary as a microreactor to perform several steps required for labeling glycans with 8-aminopyrene-1,3,6 trisulfonic acid and at the same time as a separation channel for CE of fluorescently labeled glycans. This could be achieved through careful optimization of all different steps, including sequential injections of fluorescent dye and glycan plugs, mixing by transverse diffusion of laminar flow profiles, incubation in a thermostatic zone, and finally separation and detection with CE. Such a complex sample treatment protocol for glycan labeling that is feasible thus far only in batchwise mode can now be converted into an automated and integrated protocol. Our approach was applied successfully to analyze fluorescently labeled N-linked oligosaccharides released from human immunoglobulin G and rituximab, a monoclonal antibody used for cancer treatment. We demonstrated the superiority of this in-capillary approach over the conventional in-tube protocol, with fourfold less reagent consumption and full automation without remarkable degradation of the glycan separation profile obtained by capillary electrophoresis.