Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

Demonstration of photonics-based D-band integrated localization and communication.

The terahertz spectrum has the ability to provide high-speed communication and millimeter-level resolution. As a result, terahertz-integrated sensing and communication (ISAC) has been identified as a key enabler for 6G wireless networks. This work discusses a photonics-based D-band communication system for integrated high-resolution localization and high-speed wireless communication. Our empirical results show that a communication rate of 5 Gbps over a distance of 1.5 m and location identification of the target with millimeter-level (<4m m) range resolution can be conducted simultaneously using the same signal. We also show that the error due to the thickness of the beam splitter can be eliminated, while the quantization error and the random drift errors are the limiting factors of the resolution achieved. This experimental demonstration using D-band communication indicates that terahertz ISAC can be realized for 6G networks while considering the underlying system restrictions (e.g., bandwidth limit and lens diameter).

Unprecedented phytoplankton blooms in autumn/winter in the southern Bohai Sea (China) due to high Yellow River discharge: Implications of extreme rainfall events.

The occurrence of abnormal phytoplankton blooms is one of the significant changes in coastal ecosystems due to climate change. However, the underlying mechanism of such blooms remains poorly understood due to the complexity of the system. In this study, the data from numerous observations was used to elucidate the unprecedented phytoplankton blooms in the autumn and winter of 2021 in Laizhou Bay, a typical aquaculture bay in the southern Bohai Sea of China. The abundance of phytoplankton cells increased by more than tenfold in the southern waters compared to that in the same period from 2019 to 2020. The phytoplankton bloom was first observed in winter in the Bohai Sea, with the cell abundance in the southern bay exceeding 108 cells L-1 in December 2021. The diversity and evenness of phytoplankton communities decreased in the southern area. Cerataulina pelagica was the dominant algae, comprising 69 % of the total phytoplankton in October and 99 % in December. In autumn 2021, the largest flood of the Yellow River in recent decades occurred. This was attributed to extreme rainfall events within the river basin. The input of substantial riverine nutrients played a significant role in promoting phytoplankton blooms. Correlation analysis indicated the important cumulative impact of the Yellow River on phytoplankton blooms rather than a direct short-term effect. Numerical modeling results indicated that exceptionally high Yellow River discharge in autumn could significantly affect the entire bay from autumn to the following spring. This study may contribute to understanding the abnormal phytoplankton blooms in coastal waters and provide valuable insights for environmental management in river basins and coastal waters.

Automation to Enable High-Throughput Chemical Proteomics.

Chemical proteomics utilizes small-molecule probes to covalently engage with their interacting proteins. Since chemical probes are tagged to the active or binding sites of functional proteins, chemical proteomics can be used to profile protein targets, reveal precise binding sites/mechanisms, and screen inhibitors competing with probes in a biological context. These capabilities of chemical proteomics have great potential to enable discoveries of both drug targets and lead compounds. However, chemical proteomics is limited by the time-consuming bottleneck of sample preparations, which are processed manually. With the advancement of robotics and artificial intelligence, it is now possible to automate workflows to make chemical proteomics sample preparation a high-throughput process. An automated robotic system represents a major technological opportunity to speed up advances in proteomics, open new frontiers in drug target discovery, and broaden the future of chemical biology.

Analysis of Cell Glycogen with Quantitation and Determination of Branching Using Liquid Chromatography-Mass Spectrometry.

Glycogen is a highly branched biomacromolecule that functions as a glucose buffer. It is involved in multiple diseases such as glycogen storage disorders, diabetes, and even liver cancer, where the imbalance between biosynthetic and catabolic enzymes results in structural alterations and abnormal accumulation of glycogen that can be toxic to cells. Accurate and sensitive glycogen quantification and structural determination are prerequisites for understanding the phenotypes and biological functions of glycogen under these conditions. In this research, we furthered cell glycogen characterization by presenting a highly sensitive method to measure the glycogen content and degree of branching. The method employed a novel fructose density gradient as an alternative to the traditional sucrose gradient to fractionate glycogen from cell mixtures using ultracentrifugation. Fructose was used to avoid the large glucose background, allowing the method to be highly quantitative. The glycogen content was determined by quantifying 1-phenyl-3-methyl-5-pyrazolone (PMP)-derivatized glucose residues obtained from acid-hydrolyzed glycogen using ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/QqQ-MS). The degree of branching was determined through linkage analysis where the glycogen underwent permethylation, hydrolysis, PMP derivatization, and UHPLC/QqQ-MS analysis. The new approach was used to study the effect of insulin on the glycogen phenotypes of human hepatocellular carcinoma (Hep G2) cells. We observed that cells produced greater amounts of glycogen with less branching under increasing insulin levels before reaching the cell's insulin-resistant state, where the trend reversed and the cells produced less but higher-branched glycogen. The advantage of this method lies in its high sensitivity in characterizing both the glycogen level and the structure of biological samples.

Expanding the Epitranscriptomic RNA Sequencing and Modification Mapping Mass Spectrometry Toolbox with Field Asymmetric Waveform Ion Mobility and Electrochemical Elution Liquid Chromatography.

Post-transcriptional modifications of RNA strongly influence the RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in the fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before MS identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA MS increases both coverage and throughput, while EMLC LC-MS orthogonally separates RNA molecules of different lengths and charges. The combination of the two methods offers a broadly applicable platform to improve the length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

Short-term changes in algal blooms and phytoplankton community after the passage of Super Typhoon Lekima in a temperate and inner sea (Bohai Sea) in China.

Extensive multi-species harmful algal blooms (HABs) were triggered by Super Typhoon Lekima in Laizhou Bay (Bohai Sea) in August 2019. After conducting two field cruises before and after the typhoon passage, we employed both high-performance liquid chromatography (HPLC)-pigment and microscopic methods to study the changes in the phytoplankton community and biomass. Following the passage of Lekima, the average surface salinity decreased, while dissolved inorganic nitrogen and dissolved silicate concentrations increased in the study area. The phytoplankton abundance and Chl a significantly increased after the typhoon event. Post-typhoon, the highest abundance values of Pseudo-nitzschia spp., Noctiluca scintillans, and Coscinodiscus spp. reached 106 cells/L and those of Bacillaria paxillifera, Ceratium spp., and Gymnodinium catenatum were in the order of 105 cells/L. HPLC-pigment CHEMTAX analysis showed that the biomass (Chl a) of dinoflagellates, diatoms, cryptophytes, chlorophytes, and haptophytes increased significantly after the typhoon. The increase in Chl a concentration was mainly attributable to large-sized phytoplankton, which are mostly diatoms and dinoflagellates. This study highlights that typhoons may cause HABs by introducing large amounts of freshwater and nutrients and change the phytoplankton community in a temperate and inner bay.

Glycan biomarkers of autoimmunity and bile acid-associated alterations of the human glycome: Primary biliary cirrhosis and primary sclerosing cholangitis-specific glycans.

We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.

Diet affects glycosylation of serum proteins in women at risk for cardiometabolic disease.

BACKGROUND: Glycoproteomics deals with glycoproteins that are formed by post-translational modification when sugars (like fucose and sialic acid) are attached to protein. Glycosylation of proteins influences function, but whether glycosylation is altered by diet is unknown. OBJECTIVE: To evaluate the effect of consuming a diet based on the Dietary Guidelines for Americans on circulating glycoproteins that have previously been associated with cardiometabolic diseases. DESIGN: Forty-four women, with one or more metabolic syndrome characteristics, completed an 8-week randomized controlled feeding intervention (n = 22) consuming a diet based on the Dietary Guidelines for Americans (DGA 2010); the remaining consumed a 'typical American diet' (TAD, n = 22). Fasting serum samples were obtained at week0 (baseline) and week8 (post-intervention); 17 serum proteins were chosen for targeted analyses. Protein standards and serum samples were analyzed in a UHPLC-MS protocol to determine peptide concentration and their glycan (fucosylation or sialylation) profiles. Data at baseline were used in correlational analyses; change in proteins and glycans following intervention were used in non-parametric analyses. RESULTS: At baseline, women with more metabolic syndrome characteristics had more fucosylation (total di-fucosylated proteins: p = 0.045) compared to women with a lesser number of metabolic syndrome characteristics. Dietary refined grain intake was associated with increased total fucosylation (ρ = - 0.530, p < 0.001) and reduced total sialylation (ρ = 0.311, p = 0.042). After the 8-week intervention, there was higher sialylation following the DGA diet (Total di-sialylated protein p = 0.018, poly-sialylated orosomucoid p = 0.012) compared to the TAD diet. CONCLUSIONS: Based on this study, glycosylation of proteins is likely affected by dietary patterns; higher sialylation was associated with a healthier diet pattern. Altered glycosylation is associated with several diseases, particularly cancer and type 2 diabetes, and this study raises the possibility that diet may influence disease state by altering glycosylation. CLINICAL TRIAL REGISTRATION: NCT02298725 at clinicaltrials.gov; https://clinicaltrials.gov/ct2/show/NCT02298725 .

Room temperature phosphorescence from Si-doped-CD-based composite materials with long lifetimes and high stability.

C-dot-based composites with phosphorescence have been widely reported due to their attractive potential in various applications. But easy quenching of phosphorescence induced by oxygen or instability of matrices remained a tricky problem. Herein, we reported a Si-doped-CD (Si-CD)-based RTP materials with long lifetime by embedding Si-CDs in sulfate crystalline matrices. The resultant Si-CD@sulfate composites exhibited a long lifetime up to 1.07 s, and outstanding stability under various ambient conditions. The intriguing RTP phenomenon was attributed to the C = O bond and the doping of Si element due to the fact that sulfates could effectively stabilize the triplet states of Si-CDs, thus enabling the intersystem crossing (ISC). Meanwhile, we confirmed that the ISC process and phosphorescence emission could be effectively regulated based on the heavy atom effect. This research introduced a new perspective to develop materials with regulated RTP performance and high stability.

Novel Metabolites from the Endophytic Fungus Chaetomium subaffine L01.

One natural p-terphenyl glycoside, gliocladinin C, and two furano-polyene derivatives, chaetominins A and B, were isolated from potato endophytic fungus Chaetomium subaffine. The absolute configurations of these compounds were elucidated by HR-ESI-MS, NMR, the DP4+ probabilities and electronic circular dichroism (ECD) spectra. Furthermore, gliocladinin C and chaetominin A showed cytotoxic activity against two selected human tumor cell lines (Hep-2 and HepG-2).

An iron-carbon-activated carbon and zeolite composite filter, anaerobic-aerobic integrated denitrification device for nitrogen removal in low C/N ratio sewage.

In this study, we use an anaerobic-aerobic integrated denitrification (Fe/C-ZACID) device with an iron-carbon-activated carbon and zeolite composite filter to remove nitrogen from simulated low carbon-nitrogen ratio (C/N) sewage. The impacts of dissolved oxygen (DO) level, hydraulic retention time (HRT), C/N and nitrate recirculation ratio on denitrification performance were studied. The results show that when HRT was 6 h, DO was 3 ± 0.1 mg/L, influent C/N was 3, and nitrate recirculation ratio was 100%, and removal rates of 95% for ammonia and 85% for total nitrogen (TN) were achieved. A beaker comparison test demonstrated that this synergistic denitrification system included heterotrophic denitrification, physicochemical denitrification, iron autotrophic denitrification and hydrogen autotrophic denitrification, etc. The Fe/C-ZACID device has a high-efficiency nitrogen removal effect for low C/N ratio sewage and strong shock resistance, which provides technical support and a theoretical basis for advanced denitrification of rural domestic sewage.

Relationship between leadership and work readiness in a cohort of new head nurses in China: A cross-sectional study.

To identify the relationship between leadership and work readiness in a cohort of new head nurses in China. This cross-sectional study enrolled 225 newly appointed head nurses in public tertiary hospitals in China, which were selected using convenience sampling. Data were collected using online questionnaires that included a sociodemographic characteristics form, the Nursing Managers Leadership Scale (NMLS), and the New Nurse Leaders' Job Readiness Scale (NNLJRS). IBM SPSS v.25 was used for statistical analysis. The overall mean scores of NMLS (100.50 ±â€…17.64) and NNLJRS (111.90 ±â€…15.84) of the 225 new nurse leaders were at moderate levels. The results of the Pearson correlation analysis and the hierarchical regression analysis further indicated that there was a significant positive correlation between leadership and work readiness of new head nurses (r = 0.85, P < .001), as well as charisma (ß = 0.19, P < .01), affinity (ß = 0.18, P < .01), coordination ability (ß = 0.32, P < .01), and motivational ability (ß = 0.21, P < .01) in leadership were found to be positively associated with work readiness. This study found that the leadership and work readiness of the new head nurses still needed improvement. A significant relationship was found between these 2 variables, and charisma, affinity, coordination ability, and motivational ability in the leadership ability of the new head nurses facilitated the level of work readiness. Nursing administration should create a leadership development series program focusing on the development of charisma, affinity, coordination ability, and motivational ability to support the work readiness of new nurse managers and help them with role transition.

Innovative behaviour profile and its associated factors among nurses in China: a cross-sectional study based on latent profile analysis.

OBJECTIVES: This study aimed to investigate the current status of innovative behaviours among nurses in traditional Chinese medicine (TCM) hospitals using latent profile analysis, identify potential subgroups and their population characteristics and explore factors associated with different categories. DESIGN: Cross-sectional study. SETTING: Six TCM hospitals in Anhui, China. PARTICIPANTS: From 1 April 2023 to 31 July 2023, a total of 642 registered nurses with more than 1 year of work experience were recruited from the clinical departments of six TCM hospitals using a stratified cluster sampling method. 529 valid questionnaires were recovered, presenting a validity rate of 82.40%. PRIMARY AND SECONDARY OUTCOME MEASURES: Data were collected through online surveys containing a sociodemographic questionnaire, the Nurse Innovative Behaviour Scale, the Nurse Adversity Quotient Self-Evaluation Scale and the Conditions for Work Effectiveness Questionnaire-II. Latent profile analysis was performed to identify categorisation features of nurses' innovative behaviour in TCM hospitals. Multiple logistic regression analyses were used to investigate associated factors with profile membership. RESULTS: TCM hospital nurses' innovative behaviours were mainly classified into three types of latent profiles: low innovative behaviour (35.3%), moderate innovative behaviour (48.4%) and high innovative behaviour (16.3%). The results of multiple logistic regression analyses indicated that gender, monthly income, department, hospital level, position, nurse competency level, any training attended related to TCM knowledge and skills, adversity quotient level and structural empowerment level were the influencing factors for the potential profiles. CONCLUSIONS: The innovative behaviour of nurses in TCM hospitals can be classified into three categories. Studying the heterogeneity of the innovative behaviour of nurses in TCM hospitals and its associated factors provides evidence for nursing administrators and educators to develop individualised interventions based on each latent characteristic to improve the innovative behaviour of nurses in TCM hospitals. It is of great significance to the heritage and innovative development of TCM nursing.

Protein oxidation of fucose environments (POFE) reveals fucose-protein interactions.

Cell membrane glycoproteins are generally highly fucosylated and sialylated, and post-translational modifications play important roles in the proteins' functions of signaling, binding and cellular processing. For these reasons, methods for measuring sialic acid-mediated protein-protein interactions have been developed. However, determining the role of fucose in these interactions has been limited by technological barriers that have thus far hindered the ability to characterize and observe fucose-mediated protein-protein interactions. Herein, we describe a method to metabolically label mammalian cells with modified fucose, which incorporates a bioorthogonal group into cell membrane glycoproteins thereby enabling the characterization of cell-surface fucose interactome. Copper-catalyzed click chemistry was used to conjugate a proximity labeling probe, azido-FeBABE. Following the addition of hydrogen peroxide (H2O2), the fucose-azido-FeBABE catalyzed the formation of hydroxyl radicals, which in turn oxidized the amino acids in the proximity of the labeled fucose residue. The oxidized peptides were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Variations in degree of protein oxidation were obtained with different H2O2 reaction times yielding the acquisition of spatial information of the fucose-interacting proteins. In addition, specific glycoprotein-protein interactions were constructed for Galectin-3 (LEG3) and Galectin-3-binding protein (LG3BP) illustrating the further utility of the method. This method identifies new fucose binding partners thereby enhancing our understanding of the cell glycocalyx.

Key issues in the STRICTA checklist. / 对STRICTAæ¸ å•å‡ ä¸ªå ³é”®é—®é¢˜çš„æ¢³ç†.

The STRICTA checklist is the guideline for reporting clinical trials undertaken using acupuncture intervention. As an extension of the CONSORT checklist, the STRICTA checklist facilitates the reporting quality of acupuncture clinical trials. The clinical research paradigm changes along with the development of science and technology. It is crucial to ensure whether or not the existing STRICTA checklist guides the reporting clinical trials of acupuncture now and in the future as well. This paper introduces the development and the updating procedure of the STRICTA checklist, analyzes the characteristics of utility and the limitation, and proposes several suggestions on the difficulties and challenges encountered in the implementation of the STRICTA checklist of current version so as to advance the further update and improvement.

Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids.

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.

Development and application of GlycanDIA workflow for glycomic analysis.

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from N-glycans, O-glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N-glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N-glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation.

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling.

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general activity-based arginylation profiling (ABAP) platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. ABAP has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 µg sample input, with 229 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.