Zhan Gao

RISING STAR ABSTRACTS Beyond Ensemble Measurements: Resolving Antigen Binding and Conformational Heterogeneity in Multispecific Antibodies

A Streamlined One-Pot Extraction Method Enables High-Sensitivity Top-Down Proteomic Characterization of Phospholamban from Biopsy-Scale Cardiac Tissue

Top-down proteomics (TDP) enables comprehensive characterization of intact proteoforms, but the analysis of membrane proteoforms remains challenging because of their hydrophobicity and relatively low abundance. To address these challenges, we previously developed Azo, a photocleavable surfactant that enables efficient solubilization and top-down analysis of membrane proteins. Subsequently, we established an Azo-enabled TDP method permitting the comprehensive characterization of phospholamban (PLN), a transmembrane protein that plays crucial roles in calcium handling, directly from cardiac tissue. However, the original workflow required at least 10 mg of human cardiac tissue, making it unsuitable for biopsy-scale and other sample-limited studies. Herein, we developed a streamlined Azo-enabled one-pot extraction strategy for high-sensitivity top-down analysis of PLN that reduces the required tissue input to 1 mg while preserving analytical performance. Using online liquid chromatography–tandem mass spectrometry, we achieved highly reproducible detection, quantification, and post-translational modification localization of endogenous PLN proteoforms from 1 mg of human cardiac tissue. Notably, the streamlined strategy reduced the tissue required for PLN protein extraction 10-fold while retaining the same capacity for comprehensive characterization in the one-pot extraction. Collectively, these results establish a practical strategy for TDP of PLN proteoforms from biopsy-scale and other sample-limited myocardial specimens.

Proteoforms in Disease: Biomedical Applications of Top-Down Proteomics.

The proteome is a dynamic landscape of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications (PTMs), which collectively drive biological function and disease phenotypes. Mass spectrometry (MS)-based proteomics has emerged as an essential technique for elucidating this molecular complexity. Although bottom-up proteomics enables deep protein identification and quantification through peptide-level analysis, it disrupts molecular connectivity and introduces a peptide-to-protein inference problem, which is suboptimal for proteoform analysis. Top-down proteomics (TDP) offers a complementary approach by analyzing intact proteins, preserving molecular connectivity, and enabling direct characterization and quantification of proteoforms. This capability is increasingly vital for understanding heterogeneous human diseases. Here, we review the evolving role of TDP in biomedical research, highlighting studies that revealed proteoform-level alterations, identified candidate biomarkers, and advanced our understanding of the roles of proteoforms in human diseases.