Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes

Bharath Sampadi,Harry Vrieling,Leon H F Mullenders

doi:10.3390/cells10123407

Bharath Sampadi, Harry Vrieling + Show 1 more

Open Access

https://doi.org/10.3390/cells10123407

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Journal: Cells	Publication Date: Dec 3, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: Leiden University Medical Center, Nagoya University

Abstract

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.

Highlights

Protein phosphorylation is a post-translational modification (PTM) that displays highly complex and dynamic properties with a dramatic low abundance compared to unmodified proteins
To prevent bias originating from the protein digestion step or enrichment material used, we applied identical conditions for both phosphoproteomics methods regarding the digestion of proteins with the Lys-c/Trypsin enzyme mix and enrichment for phosphopeptides using titanium dioxide (TiO2) beads
For strong cation exchange chromatography-based phosphoproteomics (SCXPhos), peptides were cleaned up using solid-phase extraction (SPE) using C18 cartridges and fractionated into 18 fractions using strong cation exchange (SCX) chromatography; subsequently, we performed phosphopeptide enrichment in each fraction using TiO2 beads to obtain phosphopeptides

Summary

Introduction

Protein phosphorylation is a post-translational modification (PTM) that displays highly complex and dynamic properties with a dramatic low abundance compared to unmodified proteins. While trypsin is highly specific and very effective in digesting proteins into peptides, it will not allow the detection of phosphopeptides that do not contain nearby R/K cleavage sites. Most phosphoproteomics studies have only used trypsin to generate phosphoproteome data while applying the 1:100 trypsin-to-peptide ratio that is standardly used for global proteomics and may have failed to detect numerous biologically important phosphosites. In line with this notion, a recent large-scale phosphoproteomics study demonstrated that the usage of multiple proteases dramatically enhances phosphopeptide identification and the sequence coverage of phosphoproteins [3]

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