Recent progress in quantitative phosphoproteomics

ABSTRACT

Introduction

Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge of dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events.

Areas covered

State-of-the-art labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization, and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples.

Expert opinion

Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in the current research, especially at single cell levels and in clinical diagnostics.

KEYWORDS:

• Isobaric labeling
• protein phosphorylation
• stable isotope labeling
• LC-MS
• quantitative phosphoproteomics
• phosphopeptide enrichment
• DIA
• hotspot thermal profiling

Article highlights

• LC-MS/MS based quantitative phosphoproteomics provides a powerful tool to analyze site-specific protein phosphorylation.

• Recent developments in phosphopeptide enrichment and labeling techniques enhance the analytic depth, dynamic range, site localization, and data integrity in quantitative phosphoproteomics.

• Advanced workflows enable comprehensive phosphoproteome analyses with less and less input material, relevant for clinical samples and single-cell analyses.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This research was funded by the Deutsche Forschungsgemeinschaft (DFG) through grant numbers GRK2364 “MOMbrane”, EXC2124 “CMFI”, FOR2816 “SCyCode”, TRR 261- Project-ID 398967434, SPP2389 “Emergent Functions of Bacterial Multicellularity” and by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 955626.