Nitric oxide inhibits ten-eleven translocation DNA demethylases to regulate 5mC and 5hmC across the genome.

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO.

Corrigendum: Glioblastoma survival is associated with distinct proteomic alteration signatures post chemoirradiation in a large-scale proteomic panel.

[This corrects the article DOI: 10.3389/fonc.2023.1127645.].

Hierarchical Self-Assembly Molecular Building Blocks as Intelligent Nanoplatforms for Ovarian Cancer Theranostics.

Hierarchical self-assembly from simple building blocks to complex polymers is a feasible approach to constructing multi-functional smart materials. However, the polymerization process of polymers often involves challenges such as the design of building blocks and the drive of external energy. Here, a hierarchical self-assembly with self-driven and energy conversion capabilities based on p-aminophenol and diethylenetriamine building blocks is reported. Through ß-galactosidase (ß-Gal) specific activation to the self-assembly, the intelligent assemblies (oligomer and superpolymer) with excellent photothermal and fluorescent properties are dynamically formed in situ, and thus the sensitive multi-mode detection of ß-Gal activity is realized. Based on the overexpression of ß-Gal in ovarian cancer cells, the self-assembly superpolymer is specifically generated in SKOV-3 cells to achieve fluorescence imaging. The photothermal therapeutic ability of the self-assembly oligomer (synthesized in vitro) is evaluated by a subcutaneous ovarian cancer model, showing satisfactory anti-tumor effects. This work expands the construction of intelligent assemblies through the self-driven cascade assembly of small molecules and provides new methods for the diagnosis and treatment of ovarian cancer.

The ESCRT protein CHMP5 promotes T cell leukemia by controlling BRD4-p300-dependent transcription.

Oncogene activity rewires cellular transcription, creating new transcription networks to which cancer cells become addicted, by mechanisms that are still poorly understood. Using human and mouse models of T cell acute lymphoblastic leukemia (T-ALL), we identify an essential nuclear role for CHMP5, a cytoplasmic endosomal sorting complex required for transport (ESCRT) protein, in establishing and maintaining the T-ALL transcriptional program. Nuclear CHMP5 promoted the T-ALL gene program by augmenting recruitment of the co-activator BRD4 by the histone acetyl transferase p300 selectively at enhancers and super-enhancers, an interaction that potentiated H3K27 acetylation at these regulatory enhancers. Consequently, loss of CHMP5 diminished BRD4 occupancy at enhancers and super-enhancers and impaired RNA polymerase II pause release, which resulted in downregulation of key T-ALL genes, notably MYC. Reinforcing its importance in T-ALL pathogenesis, CHMP5 deficiency mitigated chemoresistance in human T-ALL cells and abrogated T-ALL induction by oncogenic NOTCH1 in vivo. Thus, the ESCRT protein CHMP5 is an essential positive regulator of the transcriptional machinery promoting T-ALL disease.

Distinct functional patterns in child and adolescent bipolar and unipolar depression during emotional processing.

Accumulating evidence from functional magnetic resonance imaging studies supported brain dysfunction during emotional processing in bipolar disorder (BD) and major depressive disorder (MDD). However, child and adolescent BD and MDD could display different activation patterns, which have not been fully understood. This study aimed to investigate common and distinct activation patterns of pediatric BD (PBD) and MDD (p-MDD) during emotion processing using meta-analytic approaches. Literature search identified 25 studies, contrasting 252 PBD patients, and 253 healthy controls (HCs) as well as 311 p-MDD patients and 263 HCs. A total of nine meta-analyses were conducted pulling PBD and p-MDD experiments together and separately. The results revealed that PBD and p-MDD showed distinct patterns during negative processing. PBD patients exhibited activity changes in bilateral precuneus, left inferior parietal gyrus, left angular gyrus, and right posterior cingulate cortex while p-MDD patients showed functional disruptions in the left rectus, left triangular part of the inferior frontal gyrus, left orbital frontal cortex, left insula, and left putamen. In conclusion, the activity changes in PBD patients were mainly in regions correlated with emotion perception while the dysfunction among p-MDD patients was in the fronto-limbic circuit and reward-related regions in charge of emotion appraisal and regulation.

Design method of dual-band synchronous zoom microscope optical system based on coaxial Kohler illumination.

The intrinsic properties of the observed object are closely related to its spectral information, to extend the imaging spectral range of a continuous zoom microscope to obtain more detailed intrinsic properties of the object, this paper proposes a design method of dual-band simultaneous zoom microscope optical system based on the coaxial Koehler uniform illumination. First, the imaging principle of the dual-band simultaneous zoom microscope optical system is theoretically analyzed, and we propose to split the front fixed group of the zoom system into a collimation lens group and a converging lens group to realize the compact design of the system. Then, two different rear fixed groups are used to correct the residual aberration, and a method for solving the initial structure of the dual-band simultaneous zoom microscope optical system is proposed. Finally, a dual-band synchronous zoom microscope optical system is designed using the method proposed in this paper. The design results show that the imaging magnification of the visible (VIS) band is -0.4 to -4.0, the simultaneous imaging magnification ranges are -0.4 to -0.8 in the VIS and short-wave infrared (SWIR) bands, and the magnification difference of its simultaneous zoom imaging is less than 1.25%. In addition, the system has the advantages of good imaging quality, clever design of coaxial illumination, and compact structure, thus verifying the feasibility of the design method.

Geometric error modeling and compensation for high precision composite optical measurement systems.

Composite optical measurement systems are widely used in the field of precision measurement due to their combination of inspection with high accuracy, speed, wide range, real-time, and other advantages. Whereas errors are prevalent in measurements, in order to improve detection accuracy, the systems must be compensated for geometric errors in three-dimensional space. Aiming at the complex situation of multi-probes and multi-zooms in the composite optical measurement system, the current error modelling methods are difficult to be directly applied, so this paper establishes a unified three-dimensional volumetric error model based on the theory of multi-body system and combined with the principle of geometric optics, performs the error verification through the direct measurement method, and finally realises the compensation of geometric error in the continuous space of the whole measurement range. Eventually, the accuracy of the proposed error model and the effectiveness of the error compensation method were verified by a laser interferometer and standard objects to be measured, and the integrated geometric error of the system was decreased by 76.55%, which effectively improved the accuracy of the system. The error modelling and compensation method proposed in this paper provides a new idea for the error compensation of the zoom measurement system, and at the same time, it is universal for the measurement systems of different structures and motion forms, which can be widely used in the field of precision measurement.

Automatic aberration compensation for digital holographic microscopy based on bicubic downsampling and improved minimization of global phase gradients.

In digital holographic microscopy, aberrations caused by imperfect optical system settings can greatly affect the quantitative measurement of the target phase, so the compensation of aberrations in the distorted phase has become a key point of research in digital holographic microscopy. Here, we propose a fully automatic numerical phase aberration compensation method with fast computational speed and high robustness. The method uses bicubic downsampling to smooth the sample phase for reducing its disturbance to the background aberration fit, while reducing the computational effort of aberration compensation. Polynomial coefficients of the aberration fitting are iteratively optimized in the process of minimizing the global phase gradient by improving the phase gradient operator and constructing the loss function to achieve accurate fitting of the phase aberration. Simulation and experimental results show that the proposed method can achieve high aberration compensation accuracy without prior knowledge of the hologram recording settings or manual selection of the background area free of samples, and it is suitable for samples with moderate and relatively flat background area, which can be widely used in the quantitative analysis of biological tissues and micro and nano structures.

Abundant binary promoter switches in lineage-determining transcription factors indicate a digital component of cell fate determination.

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors in vitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.

A Membrane-Targeted Photosensitizer Prevents Drug Resistance and Induces Immune Response in Treating Candidiasis.

Candida albicans (C. albicans), a ubiquitous polymorphic fungus in humans, causes different types of candidiasis, including oral candidiasis (OC) and vulvovaginal candidiasis (VVC), which are physically and mentally concerning and financially costly. Thus, developing alternative antifungals that prevent drug resistance and induce immunity to eliminate Candida biofilms is crucial. Herein, a novel membrane-targeted aggregation-induced emission (AIE) photosensitizer (PS), TBTCP-QY, is developed for highly efficient photodynamic therapy (PDT) of candidiasis. TBTCP-QY has a high molar absorption coefficient and an excellent ability to generate 1 O2 and •OH, entering the interior of biofilms due to its high permeability. Furthermore, TBTCP-QY can efficiently inhibit biofilm formation by suppressing the expression of genes related to the adhesion (ALS3, EAP1, and HWP1), invasion (SAP1 and SAP2), and drug resistance (MDR1) of C. albicans, which is also advantageous for eliminating potential fungal resistance to treat clinical infectious diseases. TBTCP-QY-mediated PDT efficiently targets OC and VVC in vivo in a mouse model, induces immune response, relieves inflammation, and accelerates the healing of mucosal defects to combat infections caused by clinically isolated fluconazole-resistant strains. Moreover, TBTCP-QY demonstrates excellent biocompatibility, suggesting its potential applications in the clinical treatment of OC and VVC.

Design of the dual-band shared-aperture asynchronous zoom optical system using focus tunable lenses.

To meet the increasing demand of the current market and the diversity of application scenarios, combine the zoom system and the multi-band shared-aperture system, and fully harness their respective advantages, this paper proposes a dual-band shared-aperture asynchronous zoom optical system using focus tunable lenses (FTLs). To address the lack of available patents for such systems, we designed a sub-system simultaneous iterative optimization algorithm to calculate the initial structure parameters. This synchronous iterative optimization approach can strengthen the connection between sub-systems and compensate for the shortcomings of current mainstream design methods. The initial structure constructed in this way has a good performance in terms of structural stability and optimization potential. Based on these methods, we successfully designed an optical system that can work in both VIS and NIR bands, and the two sub-systems can zoom independently. The design results possess good performance in terms of distortion control, aberration correction, and volume control.

Glioblastoma survival is associated with distinct proteomic alteration signatures post chemoirradiation in a large-scale proteomic panel.

Background: Glioblastomas (GBM) are rapidly progressive, nearly uniformly fatal brain tumors. Proteomic analysis represents an opportunity for noninvasive GBM classification and biological understanding of treatment response. Purpose: We analyzed differential proteomic expression pre vs. post completion of concurrent chemoirradiation (CRT) in patient serum samples to explore proteomic alterations and classify GBM by integrating clinical and proteomic parameters. Materials and methods: 82 patients with GBM were clinically annotated and serum samples obtained pre- and post-CRT. Serum samples were then screened using the aptamer-based SOMAScan® proteomic assay. Significant traits from uni- and multivariate Cox models for overall survival (OS) were designated independent prognostic factors and principal component analysis (PCA) was carried out. Differential expression of protein signals was calculated using paired t-tests, with KOBAS used to identify associated KEGG pathways. GSEA pre-ranked analysis was employed on the overall list of differentially expressed proteins (DEPs) against the MSigDB Hallmark, GO Biological Process, and Reactome databases with weighted gene correlation network analysis (WGCNA) and Enrichr used to validate pathway hits internally. Results: 3 clinical clusters of patients with differential survival were identified. 389 significantly DEPs pre vs. post-treatment were identified, including 284 upregulated and 105 downregulated, representing several pathways relevant to cancer metabolism and progression. The lowest survival group (median OS 13.2 months) was associated with DEPs affiliated with proliferative pathways and exhibiting distinct oppositional response including with respect to radiation therapy related pathways, as compared to better-performing groups (intermediate, median OS 22.4 months; highest, median OS 28.7 months). Opposite signaling patterns across multiple analyses in several pathways (notably fatty acid metabolism, NOTCH, TNFα via NF-κB, Myc target V1 signaling, UV response, unfolded protein response, peroxisome, and interferon response) were distinct between clinical survival groups and supported by WGCNA. 23 proteins were statistically signficant for OS with 5 (NETO2, CST7, SEMA6D, CBLN4, NPS) supported by KM. Conclusion: Distinct proteomic alterations with hallmarks of cancer, including progression, resistance, stemness, and invasion, were identified in serum samples obtained from GBM patients pre vs. post CRT and corresponded with clinical survival. The proteome can potentially be employed for glioma classification and biological interrogation of cancer pathways.

Multi-omics Pathways Workflow (MOPAW): An Automated Multi-omics Workflow on the Cancer Genomics Cloud.

Introduction: In the era of big data, gene-set pathway analyses derived from multi-omics are exceptionally powerful. When preparing and analyzing high-dimensional multi-omics data, the installation process and programing skills required to use existing tools can be challenging. This is especially the case for those who are not familiar with coding. In addition, implementation with high performance computing solutions is required to run these tools efficiently. Methods: We introduce an automatic multi-omics pathway workflow, a point and click graphical user interface to Multivariate Single Sample Gene Set Analysis (MOGSA), hosted on the Cancer Genomics Cloud by Seven Bridges Genomics. This workflow leverages the combination of different tools to perform data preparation for each given data types, dimensionality reduction, and MOGSA pathway analysis. The Omics data includes copy number alteration, transcriptomics data, proteomics and phosphoproteomics data. We have also provided an additional workflow to help with downloading data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium and preprocessing these data to be used for this multi-omics pathway workflow. Results: The main outputs of this workflow are the distinct pathways for subgroups of interest provided by users, which are displayed in heatmaps if identified. In addition to this, graphs and tables are provided to users for reviewing. Conclusion: Multi-omics Pathway Workflow requires no coding experience. Users can bring their own data or download and preprocess public datasets from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium using our additional workflow based on the samples of interest. Distinct overactivated or deactivated pathways for groups of interest can be found. This useful information is important in effective therapeutic targeting.

Cohesin regulates alternative splicing.

Cohesin, a trimeric complex that establishes sister chromatid cohesion, has additional roles in chromatin organization and transcription. We report that among those roles is the regulation of alternative splicing through direct interactions and in situ colocalization with splicing factors. Degradation of cohesin results in marked changes in splicing, independent of its effects on transcription. Introduction of a single cohesin point mutation in embryonic stem cells alters splicing patterns, demonstrating causality. In primary human acute myeloid leukemia, mutations in cohesin are highly correlated with distinct patterns of alternative splicing. Cohesin also directly interacts with BRD4, another splicing regulator, to generate a pattern of splicing that is distinct from either factor alone, documenting their functional interaction. These findings identify a role for cohesin in regulating alternative splicing in both normal and leukemic cells and provide insights into the role of cohesin mutations in human disease.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

CCLOWW: A grade-level Chinese children's lexicon of written words.

In this article, we present the Chinese Children's Lexicon of Written Words (CCLOWW), the first grade-level database that provides frequency statistics of simplified Chinese characters and words for children. The database computes from a corpus of 34,671,424 character tokens and 22,427,010 word tokens (including single- and multicharacter words), extracted from 2131 books. It contains 6746 different character types and 153,079 different word types. CCLOWW provides several frequency indices of simplified Chinese for three grade levels (grade 2 and below, grades 3-4, grades 5-6) to profile children's experience with written Chinese in and outside of school. We describe in this article the distributions of frequency and contextual diversity of the characters and words, as well as word length and syntactic categories of the words in the corpus and the subcorpora. We also report results of correlation analyses with other written corpora and of several naming and lexicon decision experiments. The findings suggest that CCLOWW frequency measures correlate well with other corpora. Importantly, they could reliably predict children's and adults' naming and lexical decision performances. They could also explain variance in adults' visual word recognition, in addition to frequency measures computed in an adult corpus, indicating that early print exposure might influence readers' lexical processing later on beyond an age of acquisition effect. CCLOWW will help researchers in language processing and development as well as educators with selecting language materials appropriate for children's developmental stages. The database is freely available online at https://www.learn2read.cn/database/ .

Engineered Phage with Aggregation-Induced Emission Photosensitizer in Cocktail Therapy against Sepsis.

Sepsis, a widely recognized disease, is characterized by multiple pathogen infections. Therefore, it is imperative to develop methods that can efficiently identify and neutralize pathogen species. Phage cocktail therapy utilizes the host specificity of phages to adapt to infect resistant bacteria. However, its low sterilization stability efficiency and lack of imaging units seriously restrict its application. Here, a novel strategy combining the aggregation-induced emission photosensitizer (AIE-PS) TBTCP-PMB with phages through a nucleophilic substitution reaction between benzyl bromide and sulfhydryl groups to remove pathogenic bacteria for sepsis treatment is proposed. This strategy retains the phage's host specificity while possessing AIE-PS characteristics with a fluorescence imaging function and reactive oxygen species (ROS) for detecting and sterilizing bacteria. This synergetic strategy combining phage cocktail therapy and photodynamic therapy (PDT) shows a strong "1 + 1 > 2" bactericidal efficacy and superior performance in sepsis mouse models with good biocompatibility. Furthermore, the strategy can quickly diagnose blood infections of clinical blood samples. This simple and accurate strategy provides a promising therapeutic platform for rapid pathogen detection and point-of-care diagnosis. Moreover, it presents a new method for expanding the library of antibacterial drugs to develop new strain identification and improve infectious disease treatment, thereby demonstrating strong translational potential.

Mitochondrial Matrix Protease ClpP Agonists Inhibit Cancer Stem Cell Function in Breast Cancer Cells by Disrupting Mitochondrial Homeostasis.

Mitochondria are multifaceted organelles which are important for bioenergetics, biosynthesis and signaling in metazoans. Mitochondrial functions are frequently altered in cancer to promote both the energy and the necessary metabolic intermediates for biosynthesis required for tumor growth. Cancer stem cells (CSCs) contribute to chemotherapy resistance, relapse, and metastasis. Recent studies have shown that while non-stem, bulk cancer cells utilize glycolysis, breast CSCs are more dependent on oxidative phosphorylation (OxPhos) and therefore targeting mitochondria may inhibit CSC function. We previously reported that small molecule ONC201, which is an agonist for the mitochondrial caseinolytic protease (ClpP), induces mitochondrial dysfunction in breast cancer cells. In this study, we report that ClpP agonists inhibit breast cancer cell proliferation and CSC function in vitro and in vivo. Mechanistically, we found that OxPhos inhibition downregulates multiple pathways required for CSC function, such as the mevalonate pathway, YAP, Myc, and the HIF pathway. ClpP agonists showed significantly greater inhibitory effect on CSC functions compared with other mitochondria-targeting drugs. Further studies showed that ClpP agonists deplete NAD(P)+ and NAD(P)H, induce redox imbalance, dysregulate one-carbon metabolism and proline biosynthesis. Downregulation of these pathways by ClpP agonists further contribute to the inhibition of CSC function. In conclusion, ClpP agonists inhibit breast CSC functions by disrupting mitochondrial homeostasis in breast cancer cells and inhibiting multiple pathways critical to CSC function. Significance: ClpP agonists disrupt mitochondrial homeostasis by activating mitochondrial matrix protease ClpP. We report that ClpP agonists inhibit cell growth and cancer stem cell functions in breast cancer models by modulating multiple metabolic pathways essential to cancer stem cell function.

Design of binocular stereo vision optical system based on a single lens and a single sensor.

To reduce the number of detectors used in conventional binocular stereo cameras, while improving the measurement accuracy and compactness of the system, this paper proposes a design method for a binocular stereo vision optical system based on a single lens and a single sensor. First, based on the design principle of the traditional binocular optical system, to the best of our knowledge, a novel method of designing a framing lens array at the optical stop of the optical system is proposed to image two images on one detector simultaneously. Second, we propose a dual-frame lens array design method at the aperture stop position of the optical system that can image two images on one detector simultaneously. Then, the design principle of the method is analyzed theoretically, as well as a detailed analysis of the imaging position layout and the stray light elimination method of the dual-channel optical system. Finally, a single-lens binocular optical system with a focal length of 20 mm and a full field of view of 30° is designed using the method in this paper, and the analysis results demonstrate that the system has the advantages of good imaging quality and compact construction and provides a design idea for the design of a binocular stereo vision optical system.

Improving Accuracy in Mass Spectrometry-Based Mass Determination of Intact Heterogeneous Protein Utilizing the Universal Benefits of Charge Reduction and Alternative Gas-Phase Reactions.

In mass analysis of proteins, mass spectrometry directly measures the mass to charge ratios of ionized proteins and promises higher accuracy than that of indirect approaches measuring other physicochemical properties, provided that the charge states of detected ions are determined. Accurate mass determination of heterogeneously glycosylated proteins is often hindered by unreliable charge determination due to the insufficient resolution of signals from different charge states and inconsistency among mass profiles of ions in individual charge states. Limited charge reduction of a subpopulation of proteoforms using electron transfer/capture reactions (ETnoD/ETnoD) solves this problem by narrowing the mass distribution of examined proteoforms and preserving the mass profile of the precursor charge state in the reduced charge states. However, the limited availability of ETnoD/ETnoD function in commercial instruments limits the application of this approach. Here, utilizing a range of charge-dependent and accuracy-affecting spectral features revealed by a systematic evaluation at levels of both the ensemble and subpopulation of proteoforms based on theoretical models and experiments, we developed a limited charge reduction workflow that enables using collision-induced dissociation and higher energy collisional dissociation, two widely available reactions, as alternatives to ETnoD/ETnoD while providing adequate accuracy. Alternatively, substituting proton transfer charge reduction for ETnoD/ETnoD provides higher accuracy of mass determination. Performing mass selection in a window-sliding manner improves the accuracy and allows profiling of the whole proteoform distribution. The proposed workflow may facilitate the development of universal characterization strategies for more complex and heterogeneous protein systems.