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

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.

Keywords: data‐independent acquisition; mass spectrometry; phosphoproteomics; protein phosphorylation.

FIGURE 1

General phosphoproteomic sample preparation workflow and the principle of phosphopeptide enrichment. (a) The typical sample preparation workflow for MS‐based phosphoproteomics involves cell lysis, protein extraction, protein digestion, peptide cleanup, phosphopeptide enrichment, and sample desalting prior to LC‐MS/MS analysis. (b) Phosphopeptides are enriched by three commonly employed strategies, including IMAC, MOAC, and IP with anti‐phosphotyrosine antibodies. IMAC and MOAC exploit the electrostatic interaction between positive metal ions and the negative phosphate groups on phosphopeptides. However, competitive binding from acidic amino acids may occur at high pH, leading to non‐specific enrichment. Thus, specific enrichment can be performed in pH/acid‐controlled conditions to achieve high specificity. Under appropriate pH, carboxylic groups on acidic residues are protonated while leaving the phosphate group deprotonated in the acidic environment. Furthermore, the addition of acids, such as acetic and lactic acids, can prevent the binding of acidic amino acids. S. T. Y.: Serine (S), Threonine (T), Tyrosine (Y). E.D.: Glutamic acid (E), Aspartic Acid (D).

FIGURE 2

Miniaturized sample preparation workflows for microscale and nanoscale phosphoproteomics. (a) Mini‐analytical system consists of PTS based workflow, miniaturized LC column (25 µm I.D.), and direct sample injection system. (b) Phospho‐SISPROT workflow consists of three integrated tips for protein digestion, enrichment, and peptide desalting. (c) Tandem tip workflow consists of three tips in tandem for desalting, enrichment, and second desalting. (d) EasyPhos workflow consists of a 96‐well plate for high‐throughput digestion and enrichment. (e) R2‐P2 workflow for single‐pot solid‐phase‐enhanced sample preparation (SP3) and 96‐well format on a magnetic particle processing robot. (f) SOP‐Phos workflow consists of DDM‐coated one‐pot sample preparation from lysis to enrichment. PTS indicates phase‐transfer surfactant; SDC, sodium deoxycholate; SCX, strong cation exchange.

FIGURE 3

Summary of recent phosphoproteomic approaches reported between 2015 and 2024, July. The figure shows the performance in the sensitivity, that is, number of phosphopeptides (shown with size of circle) identified per nanogram (ng) of samples under different sample input amounts.

FIGURE 4

Schematic overview of MS‐based quantitative phosphoproteomics approaches. (a) In label‐free approaches, every sample is individually processed and subjected to LC‐MS/MS analysis. Quantitation is performed by the peak area of extracted MS¹ or MS² ion chromatograms. (b) In metabolic labeling, amino acids with heavy isotopes are added to the cell culture medium. During cell growth, these amino acids are incorporated into proteins. Consequently, proteins from the heavy isotope condition can be distinguished from their light counterparts by a fixed mass shift. Quantitation is performed by the relative intensity or extracted ion chromatogram of the isotopic pairs. (c) In chemical labeling, isotope labeling‐based tags react with peptides (or protein) and samples are mixed and injected to LC‐MS/MS in one run. Quantitation is performed by comparing the relative intensities of the isotopic reporter ions. TMT indicates tandem mass tag.