A novel workflow for quantitative phosphoproteomics analysis using HiRIEF LC-MS

Panizza E, Branca RMM, Oliviusson P, Orre LM & Lehtiö J

Scientific Reports 7, Article number: 4513 (2017), doi:10.1038/s41598-017-04798-z



Key features of the HiRIEF-based phosphoproteomics method:

- Identified and quantified 18,382 phosphorylation sites in HeLa cells
- Requires low amount of material (300 µg/sample) and allows for multiplex quantification using TMT
- Identified 1,264 novel phospho-sites 
- 6.3% of the detected phospho-sites are on tyrosine residues (1,203 pY sites)
- HiRIEF fractionation using the novel ultra-acidic pH range gel strip enriches for multiply phosphorylated peptides
- Kinase association analysis pinpointed 165 novel phospho-sites putatively functional during mitosis


In eukaryotic cells, thousands of highly regulated protein phosphorylation events operate to regulate fundamental cellular functions such as proliferation, survival, apoptosis, migration and metabolism. Deregulation of the intricate network of cellular phosphorylations is involved in different human diseases, including neurodegenerative diseases and cancer. Methods to study protein phosphorylation on a large-scale (phosphoproteomics) have emerged and have been developed for the last 15 years, but the low abundance of phosphorylated proteins in the cell makes it a challenging task.

In this work we develop a new workflow for phosphoproteomic analysis that uses isoelectric point-based peptide separation (high-resolution isoelectric focusing, HiRIEF) coupled to liquid chromatography-mass spectrometry (LC-MS). The presented workflow allows to simultaneously quantify 10 samples by employing isobaric tags (TMT) and requires a relatively low amount of starting material (300 µg of peptides per sample). 

We analyzed HeLa cells (from human cervix adenocarcinoma), either untreated, treated with a drug that increases tyrosine phosphorylation, or cell-cycle arrested in mitosis using nocodazole. We were interested in mitotic-arrested cells as they are characterized by a very high number of phosphorylation events, the majority of which have an unknown function. 

To address this issue, we conducted a bioinformatic analysis that predicts which phosphorylation sites may have a regulatory function. The analysis estimates the affinity of each phospho-site to different protein kinases: phospho-sites that are predicted to interact strongly with a protein kinase are candidates to have a regulatory function, based on the fact that a strong interaction with a protein kinase is necessary for precise phospho-regulation. With this approach we identify 165 novel, putatively functional phospho-sites. Among those, we identify for example two novel phospho-sites on the centromeric proteins CENP-C and CENP-F (represented in the figure), that might affect these proteins ability to interact with other proteins at the chromosomes' centromeres and therefore affect the process of chromosomes segregation during mitosis.

In summary, the HiRIEF-based phosphoproteomics workflow allows for in-depth profiling of the cellular phosphoproteome, compatible with multiplex quantification. Furthermore, our work provides a curated list of novel phosphorylation sites that are putatively functional during mitosis and can be used for future studies on the role of phosphorylation during mitotic progression.