Biology subjects

Wang, M.-D.

Publications and source records attributed to Wang, M.-D..

5 recordsLinked to original sources

bioRxiv2026

Integrated Multi-Omics Enabled by Sequential Extraction for Comprehensive Molecular Profiling of Small Extracellular Vesicles

Small extracellular vesicles (sEVs) are membrane-bound particles whose protein, lipid, and metabolite cargo reflects the molecular state of their cells of origin, making them attractive targets for biomarker discovery and therapeutic development. However, comprehensive characterization of sEVs remains challenging due to the extremely limited material available. Here, we present an integrated mass spectrometry-based multi-omics platform for simultaneous characterization of lipids, metabolites, and proteins from a single sEV sample enabled by sequential extraction, maximizing sample utilization. To enhance molecular coverage and analytical depth, the platform combines iterative tandem mass spectrometry for improved small-molecule fragmentation and nano-flow proteomics with data-independent acquisition. We achieved deep and reproducible multi-omic characterization of proteins, lipids, and metabolites using 10 million sEVs. We further demonstrated the compatibility of our multi-omics platform with sEVs isolated from plasma by ultracentrifugation, size-exclusion chromatography with ultrafiltration, and polymer precipitation, revealing purification-dependent differences in molecular profiles associated with tradeoffs in yield and purity of sEVs. By enabling integrated multi-omics from the same sample, this strategy addresses a key challenge in low-input sEV analysis and establishes a robust analytical foundation for synergistic biomarker discovery and therapeutic applications.

biochemistry↗

bioRxiv2025

Proteoform-Resolved Phosphorylation Dynamics in Kinase Complexes by Hybrid Precision Mass Spectrometry

Protein kinases integrate cellular signals through complex phosphorylation cascades, yet resolving how chemical perturbations trigger and modulate these cascades in therapeutic targets remains a major challenge. Here, we dissect AMP-activated protein kinase (AMPK) proteoforms during activation through controlled biochemical reactions with a hybrid mass spectrometry (MS) approach integrating bottom-up MS for site-specific kinetics with top-down proteoform characterization. We reveal that AMPK phosphorylation proceeds through hierarchical cascades rather than binary switching, with dual entry points: canonical CaMKK2-mediated phosphorylation or allosteric activator PF-739 both triggering extensive autophosphorylation with 1-S496 showing highest kinetic priority. Proteoform-resolved analysis uncovers channeled {beta}1-S24/25+S108 co-phosphorylation linking subcellular localization with allosteric responsiveness. Site-directed mutagenesis demonstrates CaMKK2 targets only 1-T183, with all other modifications arising through autophosphorylation. Phosphatase competition reveals asymmetric control where PP1A selectively removes activation-loop phosphorylation while autophosphorylation sites remain protected, establishing persistent regulatory states. Resolving AMPKs temporal kinetics and proteoform architecture during activation enables a proteoform-centric understanding on kinase regulation.

biochemistry↗

bioRxiv2025

Structural Heterogeneity of Proteoform-Ligand Complexes in AMP-Activated Protein Kinase Uncovered by Integrated Top-Down Mass Spectrometry

AMP-activated protein kinase (AMPK) is a heterotrimeric complex ({beta}{gamma}) that serves as a master regulator of cellular metabolism, making it a prominent drug target for various diseases. Post-translational modifications (PTMs) and ligand binding significantly affect the activity and function of AMPK. However, the dynamic interplay of PTMs, non-covalent interactions, and higher-order structures of the kinase complex remains poorly understood. Herein, we report the structural heterogeneity of the AMPK complex arising from ligand binding and proteoforms--protein products derived from PTMs, alternative splicing, and genetic variants--using integrated native and denatured top-down mass spectrometry (TDMS). The fully intact AMPK heterotrimeric complex exhibits heterogeneity due to phosphorylation and multiple adenosine monophosphate (AMP) binding states. Native TDMS delineates the subunit composition, AMP binding stoichiometry, and higher-order structure of AMPK complex, whilst denatured TDMS comprehensively characterizes the proteoforms and localizes the phosphorylation site. This is the first study to structurally characterize AMPK proteoform-ligand complexes. Notably, by integrating native TDMS and AlphaFold, we elucidate a flexibly connected regulatory region of AMPK {beta} subunit that has been difficult to visualize with traditional structural biology tools. Our findings uncover previously unresolvable structural features of AMPK, offer new perspectives on protein kinase regulation, and establish a versatile framework for comprehensive characterization of proteoform-ligand complexes.

biochemistry↗

bioRxiv2023

Native nanoproteomics captures the structure and dynamics of endogenous protein complexes in human heart tissue

Protein complexes are highly dynamic entities that display substantial diversity in their assembly, post-translational modifications, and non-covalent interactions, allowing them to play critical roles in various biological processes. The heterogeneity, dynamic nature, and low abundance of protein complexes in their native states present tremendous challenges to study using conventional structural biology techniques. Here we develop a "native nanoproteomics" strategy for the native enrichment and subsequent native top-down mass spectrometry (nTDMS) of of low-abundance protein complexes. Specifically, we demonstrate the first comprehensive characterization of the structure and dynamics of cardiac troponin (cTn) complexes directly from human heart tissue. The endogenous cTn complex is effectively enriched and purified using peptide-functionalized superparamagnetic nanoparticles under non-denaturing conditions to enable the isotopic resolution of cTn complexes, revealing their complex structure and assembly. Moreover, nTDMS elucidates the stoichiometry and composition of the heterotrimeric cTn complex, localizes Ca2+ binding domains (II-IV), defines cTn-Ca2+ binding dynamics, and provides high-resolution mapping of the proteoform landscape. This native nanoproteomics strategy opens a new paradigm for structural characterization of low-abundance native protein complexes.

biochemistry↗

bioRxiv2019

The Relationship between Parasitic Fleas and Small Mammals in Household of Western Yunnan Province, China

The Yunnan Province has one of the most serious outbreaks of the plague epidemic in China. Small mammals and fleas are risk factors for the occurrence of plague in commensal plague foci. Understanding the relationship between parasitic fleas and small mammals will help control fleas and prevent the onset of the plague. Four hundred and twenty-one small mammals, belonging to 9 species, were captured. Of these, 170 small mammals (40.4%) were infested with fleas. A total of 992 parasitic fleas (including 5 species) was collected. The number of Leptopsylla Segnis and Xenopsylla Cheopis was 91.0%. The final multiple hurdle negative binomial regression model showed that when compared with Rattus Tanezumi, the probability of flea infestation on Mus musculus and other host species decreased from 58% to 99%, while the infestation with fleas from other host species increased 4.7 fold. The probability of flea prevalence in adult hosts increased by 74%, while the number of fleas decreased by 76%. The number of flea infestations in small male mammals increased by 62%. The number of fleas in small mammals weighing more than 59 grams has been multiplied by about 4. Rattus Tanezumi is the predominant species in households in West Yunnan Province, while Leptopsylla Segnis and Xenopsylla Cheopis are dominant parasitic fleas. There is a strong relationship between the abundance of parasitic fleas and the characteristics of small mammals (e.g. Species, age, sex, and body weight).

zoology↗