Single-Cell Proteomics using Ultrahigh-Resolution Mass Spectrometry to Unravel Cellular Heterogeneity in Complex Tissues

Understanding cellular heterogeneity is critical for deciphering the complexity of biological systems, disease mechanisms, and therapeutic responses. Traditional bulk proteomics, while valuable, masks the diversity among individual cells by averaging protein expression profiles across heterogeneous populations. This limitation impedes insights into rare cell types, transient cell states, and functional subpopulations within complex tissues such as tumors, the brain, and the immune system. The emergence of single-cell proteomics (SCP) offers a transformative approach to profile proteins at single-cell resolution, thereby enabling high-definition molecular phenotyping. Ultrahigh-resolution mass spectrometry (UHR-MS) has emerged as a pivotal technology in advancing SCP by allowing the detection of low-abundance proteins, resolving isobaric species, and improving the dynamic range of quantification. Recent innovations in sample preparation, including nanodroplet processing, ultra-low-flow liquid chromatography, and ion mobility spectrometry, have significantly enhanced proteome coverage and reproducibility in single-cell analyses. These advances now permit the quantification of thousands of proteins per cell, shedding light on functional diversity that underpins tissue organization and disease progression. This study highlights the integration of UHR-MS in SCP workflows to dissect cellular heterogeneity in complex tissues. We explore its application in profiling immune cell subtypes in inflamed tissues, mapping cancer cell plasticity, and tracking lineage-specific proteomic signatures in neurodevelopment. The review also discusses current challenges such as throughput, sensitivity, and data interpretation, and presents future directions, including machine learning-assisted analysis and multiplexed approaches. Single-cell proteomics powered by UHR-MS stands poised to redefine our understanding of cell biology in health and disease.