Biology subjects

Chan, H.-J.

Publications and source records attributed to Chan, H.-J..

5 recordsLinked to original sources

bioRxiv2026

An Integrated Experimental and Informatics Strategy Enables Large Proteoform Identification in Top-Down Proteomics

Top-down proteomics enables identification and characterization of intact proteoforms, preserving information on co-occurring sequence variations and post-translational modifications. However, identifying large proteoforms remains challenging due to an exponential decrease in signal-to-noise ratio with increasing molecular weight, interference from coeluting smaller species, unresolved precursor isotopic envelopes, difficulty obtaining informative MS/MS spectra, and limitations of current informatics tools. To address these challenges, here we present an integrated experimental and informatics strategy combining protein separation, data acquisition, precursor mass assignment, and MS/MS interpretation. First, we demonstrate that online two-dimensional liquid chromatography coupling size-exclusion and reversed-phase separations enables facile detection of large proteoforms. We then utilize a precursor mass correction strategy to enable conventional database searching of data-dependent acquisition (DDA) data containing isotopically unresolved precursors. Finally, we use data-independent acquisition (DIA) optimized for large proteins to enhance fragment signal and enable identification through intact-mass-guided candidate discovery and MS/MS validation. Applied to human heart tissue, the integrated strategy identified 703 nonredundant proteoforms from 236 unique proteins, including 65 proteins >30 kDa and 22 proteins >50 kDa, with identifications extending to 223 kDa. This integrated strategy bridges the detection-to-identification gap for large proteins and expands the accessible mass range of discovery top-down proteomics.

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↗

bioRxiv2024

In-depth Characterization of S-Glutathionylation in Ventricular Myosin Light Chain 1 Across Species by Top-Down Proteomics

S-glutathionylation (SSG) is increasingly recognized as a critical signaling mechanism in the heart, yet SSG modifications in cardiac sarcomeric proteins remain understudied. Here we identified SSG of the ventricular isoform of myosin light chain 1 (MLC-1v) in human, swine, and mouse cardiac tissues using top-down mass spectrometry (MS)-based proteomics. Our results enabled the accurate identification, quantification, and site-specific localization of SSG in MLC-1v across different species. Notably, the endogenous SSG of MLC-1v was observed in human and swine cardiac tissues but not in mice. Treating non-reduced cardiac tissue lysates with GSSG elevated MLC-1v SSG levels across all three species.

biochemistry↗

bioRxiv2023

MASH Native: A Unified Solution for Native Top-Down Proteomics Data Processing

Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

bioinformatics↗