Chemical Labeling Research Articles

Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides.

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

Regio‐ and Stereoselective Synthesis of Ynenamides through Gold(I)‐Catalyzed Hydroalkynylation of Ynamides

AbstractTransition metal catalysis is a useful tool for C−C bond‐forming reactions. Of special interest is the hydroalkynylation reaction of alkynes, which provides a simple method for the synthesis of enynes representing interesting structural motifs in organic chemistry. So far, there are only a few examples of gold(I)‐catalyzed hydroalkynylation reactions. Herein, we were able to show experimentally that the gold(I)‐catalyzed hydroalkynylation of ynamides leads regio‐ and stereoselectively to the corresponding ynenamides, a rather unknown type of molecule. A wide range of products could be generated with yields up to 90 % and, in contrast to many other gold‐catalyzed alkyne dimerization reactions, alkyl groups are tolerated. Quantum chemical calculations and NMR labelling experiments reveal a mechanism via dual gold catalysis.

Metabolomic Profiling Reveals Changes in Amino Acid and Energy Metabolism Pathways in Liver, Intestine and Brain of Zebrafish Exposed to Different Thermal Conditions

Global warming is predicted to increase prolonged thermal challenges for aquatic ectotherms, i.e. it causes metabolic performance declines, impacts food intake, and finally causes impaired growth. In this research work, we investigated whether a tropical fish, Danio rerio (zebrafish), could tolerate prolonged thermal challenges and whether the temperature increase has a significant impact on growth and metabolism. To answer our questions, we evaluate the metabolomic performance, a question that has received little attention so far, using differential chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS). Three groups of fish were exposed to various temperatures of 27.6 ± 2°C, 30.7 ± 2°C or 32.2 ± 2°C during 270 days post fecundation (dpf) to evaluate the impact of the temperature increase on the growth and metabolomic performance. The results obtained demonstrated different metabolomic changes in response to acclimation to the different temperatures. After 270 days, the fish maintained at the highest tested temperature (32°C) showed reduced growth, reduced condition factor, and elevated levels of metabolites associated with amino acid catabolism and lipid metabolism pathways in the liver and intestine compared with fish kept at lower temperatures (27.6 ± 2°C). These findings demonstrate an explicit redistribution of energy stores and protein catabolism in fish at the highest temperature, thus showing a preference for maintaining length growth during limited energy availability. Moreover, here we also screened out both the marker metabolites and the altered metabolic pathways to provide essential insights to ascertain the effects of the water temperature increase on the growth and development of tropical fish.

Screening and Identification of Potential Abscisic Acid Catabolites by Chemical Labeling-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry.

In this study, a screening strategy was established based on ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry assisted by chemical isotope labeling (CIL-UPLC-HRMS) for screening and identifying abscisic acid (ABA) catabolites. Based on the structures of known ABA catabolites, this strategy first proposed the structures of catabolites to be discovered. Afterward, a pair of isotope reagents N,N-2-dimethylaminoethylamine (DMED) and d4-DMED were used as labeling reagents to label the carboxyl groups in ABA and its catabolites. Then, the mass-to-charge ratio (m/z) of DMED- and d4-DMED-labeled ABA catabolites was calculated based on the labeling schema. In light of the characteristic fragmentation patterns of the DMED-labeled standards of ABA and its catabolites, screening criteria were formulated. Using our strategy, ABA, t-ABA, and 18 ABA catabolites were identified from seven plant samples. Of the identified catabolites, 16 were known, and to our knowledge, 2 were previously unidentified. Our findings contribute to ABA catabolic network improvement.

Development of a High-Coverage Quantitative Metabolome Analysis Method Using Four-Channel Chemical Isotope Labeling LC-MS for Analyzing High-Salt Fermented Food.

Metabolome analysis of high-salt fermented food can be an analytical challenge, as the salts can interfere with the sample processing and analysis. In this work, we describe a four-channel chemical isotope labeling (CIL) LC-MS approach for a comprehensive metabolome analysis of high-salt fermented food. The workflow includes metabolite extraction, chemical labeling of metabolites using dansyl chloride, dansylhydrazine, or p-dimethylaminophenacyl bromide reagents to enhance separation and ionization, LC-UV measurement of the total concentration of dansyl-labeled metabolites in each sample for sample normalization, mixing of 13C- and 12C-reagent-labeled samples, high-resolution LC-MS analysis, and data processing. Metabolome analysis of fermented foods, including fermented red pepper (FRP) sauce, soy sauce, and sufu (a fermented soybean food), showed unprecedented high metabolic coverage. Metabolome comparison of FRP, soy sauce, and sufu, as well as soy sauce and sufu, indicated great diversity of metabolite types and abundances in these foods. In addition, we analyzed two groups of samples of the same type, FRP with 10% (w/w) and 15% (w/w) salt contents, and detected large variations in multiple categories of metabolites belonging to a number of different metabolic pathways. We envisage that this CIL LC-MS approach can be generally used for metabolomic studies of high-salt fermented food. CIL LC-MS allows high-coverage identification and quantification that could not be done using other methods.

Resolving m6A epitranscriptome with stoichiometry

A recent study by Hu et al. describes N6-methyladenosine (m6A)-selective allyl chemical labeling and sequencing (m6A-SAC-seq), which allows for quantitative, stoichiometric, and positional analyses of m6A at single-nucleotide resolution across the whole transcriptome level. Information on the m6A stoichiometry will provide additional layers of gene regulatory pathways mediated by m6A modification during diverse molecular, cellular, and physiological events.

Photophysical Properties of Fluorescent Labels: A Meta‐Analysis to Guide Probe Selection Amidst Challenges with Available Data

AbstractFluorescent labels have been paramount in the advancement of molecular biology and related fields, for they unveil Nature's unknowns. Numerous labels have been developed that can be attached to most biomolecules via various labeling chemistries. Practitioners are expected to navigate through the growing libraries of fluorescent probes to select the best one for their target application. It is precisely this vast collection of labels that makes the selection process challenging, often leading to non‐ideal choices. Here, we present a meta‐analysis of fluorescent labels data with the intention of facilitating the selection process. We classify tags into molecular (dyes), macromolecular (proteins), and nanoparticles. Groups are then organised into subgroups by chemical structure. Entries were structured based on cost, absorption and emission wavelengths, Stokes shifts, molar extinction coefficients, quantum yields, lifetimes, and brightness. We correlate chemical composition to available photophysical properties of the fluorophores in their bound‐whenever possible‐and unbound states. The limited access to complete, comparable, and meaningful photophysical data for fluorescent labels and to reliable fluorescence standards, where calibration is still challenging and benchmarks are still ill‐defined, are highlighted to raise critical awareness of the current issues hampering further development of the field.

Multiplex Chemical Labeling of Amino Acids for Protein Footprinting Structure Assessment.

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.

Biocapture-Directed Chemical Labeling for Discerning Stressed States of Organelles.

Lysosomal rupture engaged in diverse diseases remains poorly discerned from lysosomal membrane permeabilization (LMP). We herein reported biocapture-directed chemical labeling (BCCL) for the discern of lysosomal rupture by tracking the release of optically labeled cathepsins from damaged lysosomes into the cytosol. BCCL entails covalent anchoring of an azide-tagged suicide substrate (Epo-LeuTyrAz) to the enzyme active site and bioorthogonal ligation of the introduced azide with DBCORC, a ratiometric sensor featuring an acidity-reporting red emissive X-rhodamine-lactam (ROX), blue emissive coumarin (CM) inert to pH, and DBCO reactive to azide. Aided with fluorescein isocyanate-labeled sialic acid (FITC-Sia), a probe remained in pH-elevated lysosomes but dissipated from LMP+ lysosomes, BCCL enables optical discern of four states of lysosomes: ruptured lysosomes (blue in cytosol), LMP+ lysosomes (blue in lysosomes), pH-elevated lysosomes (blue and green in lysosomes), and physiological lysosomes (blue, green and red in lysosomes). This approach could find applicability to study lysosome rupture over LMP in diseases and to evaluate lysosome rupture-inducing drugs.

29‐Plex tandem mass tag mass spectrometry enabling accurate quantification by interference correction

Tandem mass tag (TMT) mass spectrometry is a mainstream isobaric chemical labeling strategy for profiling proteomes. Here we present a 29-plex TMT method to combine the 11-plex and 18-plex labeling strategies. The 29-plex method was examined with a pooled sample composed of 1×, 3×, and 10× Escherichia coli peptides with 100× human background peptides, which generated two E. coli datasets (TMT11 and TMT18), displaying the distorted ratios of 1.0:1.7:4.2 and 1.0:1.8:4.9, respectively. This ratio compression from the expected 1:3:10 ratios was caused by co-isolated TMT-labeled ions (i.e., noise). Interestingly, the mixture of two TMT sets produced MS/MS spectra with unique features for the noise detection: (i) in TMT11-labeled spectra, TMT18-specific reporter ions (e.g., 135N) were shown as the noise; (ii) in TMT18-labeled spectra, the TMT11/TMT18-shared reporter ions (e.g., 131C) typically exhibited higher intensities than TMT18-specific reporter ions, due to contaminated TMT11-labeled ions in these shared channels. We further estimated the noise levels contributed by both TMT11- and TMT18-labeled peptides, and corrected reporter ion intensities in every spectrum. Finally, the anticipated 1:3:10 ratios were largely restored. This strategy was also validated using another 29-plex sample with 1:5 ratios. Thus the 29-plex method expands the TMT throughput and enhances the quantitative accuracy.

Dihydrouridine in the Transcriptome: New Life for This Ancient RNA Chemical Modification.

Until recently, post-transcriptional modifications of RNA were largely restricted to noncoding RNA species. However, this belief seems to have quickly dissipated with the growing number of new modifications found in mRNA that were originally thought to be primarily tRNA-specific, such as dihydrouridine. Recently, transcriptomic profiling, metabolic labeling, and proteomics have identified unexpected dihydrouridylation of mRNAs, greatly expanding the catalog of novel mRNA modifications. These data also implicated dihydrouridylation in meiotic chromosome segregation, protein translation rates, and cell proliferation. Dihydrouridylation of tRNAs and mRNAs are introduced by flavin-dependent dihydrouridine synthases. In this review, we will briefly outline the current knowledge on the distribution of dihydrouridines in the transcriptome, their chemical labeling, and highlight structural and mechanistic aspects regarding the dihydrouridine synthases enzyme family. A special emphasis on important research directions to be addressed will also be discussed. This new entry of dihydrouridine into mRNA modifications has definitely added a new layer of information that controls protein synthesis.

Recent advances in isobaric labeling and applications in quantitative proteomics.

Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.

Dibenzocyclooctendiones (DBCDOs): Arginine-Selective Chemical Labeling Reagents Obtained through Benzilic Acid Rearrangement.

We demonstrate that dibenzocyclooctendiones (DBCDOs) are efficient chemical reagents for the site-specific labeling of arginine-containing biomolecules. Unlike the commonly used probes, DBCDOs undergo an irreversible ring-contracted rearrangement with the guanidinium group on arginine residues under mild reaction conditions. The regioselective dual-labeled arginine residues were obtained in a one-pot reaction with our tested substrates. The efficiency of DBCDOs reactions and their ease of synthesis make DBCDOs an attractive choice for the site-selective bioconjugation of arginine.

Label-free detection and enumeration of Giardia cysts in agitated suspensions using in situ microscopy

Laboratory procedures performed in water treatment studies frequently require the characterization of (oo)cyst suspensions. Standard methods commonly used are laborious, expensive and time-consuming, besides requiring well-trained personnel to prepare samples with fluorescent staining and perform analysis under fluorescence microscopy. In this study, an easy cost-effective in situ microscope was assessed to acquire images of Giardia cysts directly from agitated suspensions without using any chemical labels or sample preparation steps. An image analysis algorithm analyzes the acquired images, and automatically enumerates and provides morphological information of cysts within 10 min. The proposed system was evaluated at different cyst concentrations, achieving a limit of detection of ~30 cysts/mL. The proposed system overcomes cost, time and labor demands by standard methods and has the potential to be an alternative technique for the characterization of Giardia cyst suspensions in resource-limited facilities, since it is independent of experts and free of consumables.

Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics

Isobaric chemical tag labels (e.g., iTRAQ and TMT) have been extensively utilized as a standard quantification approach in bottom-up proteomics, which provides high multiplexing capacity and enables MS2-level quantification while not complicating the MS1 scans. We recently demonstrated the feasibility of intact protein TMT labeling for the identification and quantification with top-down proteomics of smaller intact proteoforms (<35 kDa) in complex biological samples through the removal of large proteins prior to labeling. Still, the production of side products during TMT labeling (i.e., incomplete labeling or labeling of unintended residues) complicated the analysis of complex protein samples. In this study, we systematically evaluated the protein-level TMT labeling reaction parameters, including TMT-to-protein mass ratio, pH/concentration of quenching buffer, protein concentration, reaction time, and reaction buffer. Our results indicated that: (1) high TMT-to-protein mass ratio (e.g., 8:1, 4:1), (2) high pH/concentration of quenching buffer (pH > 9.1, final hydroxylamine concentration >0.3%), and (3) high protein concentration (e.g., > 1.0 μg/μL) resulted in optimal labeling efficiency and minimized production of over/underlabeled side products. >90% labeling efficiency was achieved for E. coli cell lysate after optimization of protein-level TMT labeling conditions. In addition, a double labeling approach was developed for efficiently labeling limited biological samples with low concentrations. This research provides practical guidance for efficient TMT labeling of complex intact protein samples, which can be readily adopted in the high-throughput quantification top-down proteomics.

RanDepict: Random chemical structure depiction generator

The development of deep learning-based optical chemical structure recognition (OCSR) systems has led to a need for datasets of chemical structure depictions. The diversity of the features in the training data is an important factor for the generation of deep learning systems that generalise well and are not overfit to a specific type of input. In the case of chemical structure depictions, these features are defined by the depiction parameters such as bond length, line thickness, label font style and many others. Here we present RanDepict, a toolkit for the creation of diverse sets of chemical structure depictions. The diversity of the image features is generated by making use of all available depiction parameters in the depiction functionalities of the CDK, RDKit, and Indigo. Furthermore, there is the option to enhance and augment the image with features such as curved arrows, chemical labels around the structure, or other kinds of distortions. Using depiction feature fingerprints, RanDepict ensures diversely picked image features. Here, the depiction and augmentation features are summarised in binary vectors and the MaxMin algorithm is used to pick diverse samples out of all valid options. By making all resources described herein publicly available, we hope to contribute to the development of deep learning-based OCSR systems.Graphical

PSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

SummaryCellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

Prosit-TMT: Deep Learning Boosts Identification of TMT-Labeled Peptides.

The prediction of fragment ion intensities and retention time of peptides has gained significant attention over the past few years. However, the progress shown in the accurate prediction of such properties focused primarily on unlabeled peptides. Tandem mass tags (TMT) are chemical peptide labels that are coupled to free amine groups usually after protein digestion to enable the multiplexed analysis of multiple samples in bottom-up mass spectrometry. It is a standard workflow in proteomics ranging from single-cell to high-throughput proteomics. Particularly for TMT, increasing the number of confidently identified spectra is highly desirable as it provides identification and quantification information with every spectrum. Here, we report on the generation of an extensive resource of synthetic TMT-labeled peptides as part of the ProteomeTools project and present the extension of the deep learning model Prosit to accurately predict the retention time and fragment ion intensities of TMT-labeled peptides with high accuracy. Prosit-TMT supports CID and HCD fragmentation and ion trap and Orbitrap mass analyzers in a single model. Reanalysis of published TMT data sets show that this single model extracts substantial additional information. Applying Prosit-TMT, we discovered that the expression of many proteins in human breast milk follows a distinct daily cycle which may prime the newborn for nutritional or environmental cues.

Comprehensive Metabolomic Comparison of Five Cereal Vinegars Using Non-Targeted and Chemical Isotope Labeling LC-MS Analysis.

Vinegar is used as an acidic condiment and preservative worldwide. In Asia, various black vinegars are made from different combinations of grains, such as Sichuan bran vinegar (SBV), Shanxi aged vinegar (SAV), Zhenjiang aromatic vinegar (ZAV), and Fujian Monascus vinegar (FMV) in China and Ehime black vinegar in Japan (JBV). Understanding the chemical compositions of different vinegars can provide information about nutritional values and the quality of the taste. This study investigated the vinegar metabolome using a combination of GC-MS, conventional LC-MS, and chemical isotope labeling LC-MS. Different types of vinegar contained different metabolites and concentrations. Amino acids and organic acids were found to be the main components. Tetrahydroharman-3-carboxylic acid and harmalan were identified first in vinegar. Various diketopiperazines and linear dipeptides contributing to different taste effects were also detected first in vinegar. Dipeptides, 3-phenyllactic acid, and tyrosine were found to be potential metabolic markers for differentiating vinegars. The differently expressed pathway between Chinese and Japanese vinegar was tryptophan metabolism, while the main difference within Chinese vinegars was aminoacyl-tRNA biosynthesis metabolism. These results not only give insights into the metabolites in famous types of cereal vinegar but also provide valuable knowledge for making vinegar with desirable health characteristics.

223 Proteomic Analysis Reveals Differential Tissue Responses to Road Transportation in Beef Steers

Abstract Thirty-six Angus-cross steers (324 ± 36 kg) were used to assess the effects of long-duration road transport on protein profiles of liver and skeletal muscle. Steers were randomly assigned to treatments (initiated on d 0): control (CON) = no transit with ad libitum access to feed, deprivation (DEPR) = no transit with no feed or water for 18 h, or transit (TRANS) = road transport with no feed or water for 18 h (1,790 km). Liver and muscle (longissimus dorsi) samples were collected from all steers immediately after treatment termination. Following extraction for total metabolite analysis, tissue protein fractions were submitted to the Protein Facility at Iowa State University for protein identification via liquid chromatography-mass spectrometry and quantification via chemical labeling with isobaric mass tags. Data were analyzed with MetaboAnalystR package v.3.0. Proteins were considered differentially abundant if the t-test P-value was ≤ 0.1 and the absolute value of change between treatments (fold change) was ≥ 1.2. In the liver, several proteins involved in energy metabolism were less abundant for DEPR or TRANS compared with CON. Nicotinate-nucleotide pyrophosphorylase, an enzyme involved in NAD biosynthesis, was less abundant in TRANS compared with CON or DEPR. Several proteins involved in inflammation and innate immunity were more abundant in TRANS compared with CON. In the muscle, alpha-2-HS-glycoprotein, a regulator of inflammation and insulin resistance, was more abundant in TRANS compared with CON or DEPR. Several proteins involved in antioxidant defense (Mn-superoxide dismutase and glutathione transferase) were less abundant in TRANS compared with DEPR. These data suggest some, but not all, physiological changes associated with long-duration transport can be attributed to feed deprivation. The physical exertion required during transport appears to affect oxidative stress in the muscle, which could impact local and systemic inflammation and nutrient metabolism.