Advancements in Global Phosphoproteomics Profiling: Overcoming Challenges in Sensitivity and Quantification

• Published in: Proteomics (Weinheim) Vol. 25; no. 1-2; pp. e202400087 - n/a
• Main Authors: Muneer, Gul, Chen, Ciao‐Syuan, Chen, Yu‐Ju
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.01.2025; John Wiley and Sons Inc
• Subjects: Animals; Biomedical materials; data‐independent acquisition; Depth profiling; Functionals; Humans; Innovations; Mapping; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Peptide mapping; Phenotypes; Phosphoproteins - analysis; Phosphoproteins - metabolism; phosphoproteomics; Phosphorylation; protein phosphorylation; Proteins; Proteome - analysis; Proteomics - methods; Review; Sample preparation; Single-Cell Analysis - methods; phosphoproteomics; mass spectrometry; data‐independent acquisition; protein phosphorylation
• ISSN: 1615-9853; 1615-9861; 1615-9861
• DOI: 10.1002/pmic.202400087

ABSTRACT Protein phosphorylation introduces post‐genomic diversity to proteins, which plays a crucial role in various cellular activities. Elucidation of system‐wide signaling cascades requires high‐performance tools for precise identification and quantification of dynamics of site‐specific phosphorylation events. Recent advances in phosphoproteomic technologies have enabled the comprehensive mapping of the dynamic phosphoproteomic landscape, which has opened new avenues for exploring cell type‐specific functional networks underlying cellular functions and clinical phenotypes. Here, we provide an overview of the basics and challenges of phosphoproteomics, as well as the technological evolution and current state‐of‐the‐art global and quantitative phosphoproteomics methodologies. With a specific focus on highly sensitive platforms, we summarize recent trends and innovations in miniaturized sample preparation strategies for micro‐to‐nanoscale and single‐cell profiling, data‐independent acquisition mass spectrometry (DIA‐MS) for enhanced coverage, and quantitative phosphoproteomic pipelines for deep mapping of cell and disease biology. Each aspect of phosphoproteomic analysis presents unique challenges and opportunities for improvement and innovation. We specifically highlight evolving phosphoproteomic technologies that enable deep profiling from low‐input samples. Finally, we discuss the persistent challenges in phosphoproteomic technologies, including the feasibility of nanoscale and single‐cell phosphoproteomics, as well as future outlooks for biomedical applications.