Abstract

Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5–3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.

Highlights

  • Protein phosphorylation is a fundamental regulatory mechanism in eukaryotic cells, affecting various processes such as cell growth, proliferation, survival, migration and metabolism

  • We explore how high-resolution isoelectric focusing (HiRIEF) liquid chromatography tandem mass spectrometry (LC-MS) performs in a quantitative phosphoproteomics workflow

  • In order to investigate how this method performs in a quantitative phosphoproteomics setting, we combined it with titanium dioxide based phospho-peptide enrichment and isobaric labeling (Fig. 1)

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Summary

Introduction

Protein phosphorylation is a fundamental regulatory mechanism in eukaryotic cells, affecting various processes such as cell growth, proliferation, survival, migration and metabolism. Current methods include reduction of sample complexity both by extensive sample fractionation and by specific phospho-peptide enrichment strategies[5] These approaches most often demand long MS-analysis time and high amount of starting material, influencing throughput and cost of the analysis, and may not even be feasible in cases where the availability of starting material is limited. Especially SILAC, have been broadly employed for phosphoproteomics analysis[5], but their multiplexing capability is limited to two or three samples. Isobaric labels, such as Tandem Mass Tags (TMT), allow simultaneous quantification of up to ten samples. Functionality of such novel phosphorylation sites was predicted to indicate sites with putative biological functions

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