Chapter 16
Mass Spectrometry-Based Proteomics for Analysis
of Hydrophilic Phosphopeptides
Chia-Feng Tsai, Jeffrey S. Smith, Dylan S. Eiger, Kendall Martin, Tao Liu,
Richard D. Smith, Tujin Shi, Sudarshan Rajagopal, and Jon M. Jacobs
Abstract
Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks
being tightly regulated by protein phosphorylation. Despite recent technological advances in reversedphase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic
phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol
was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide
hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of
identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method,
we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor
(GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This
protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis
of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.
Key words Hydrophilic phosphopeptide, Phosphoproteomics, TMT labeling, Phosphopeptide
enrichment, Mass spectrometry
1 Introduction
Cell signaling is highly regulated by protein phosphorylation [1–
3]. Changes in phosphorylation stoichiometry are used as an indicator of signaling pathway activation (cellular functional states) [1–
3]. Aberrant protein phosphorylation is linked to human diseases
including cancer [2–4]. Mass spectrometry (MS)-based phosphoproteomics has emerged as a powerful tool for comprehensive, sitespecific, quantitative phosphoproteome profiling. Due to the low
stoichiometry of protein phosphorylation and low ionization efficiency of phosphopeptides, enrichment of phosphopeptides (e.g.,
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
247
Mass Spectrometry-Based Proteomics for Analysis
of Hydrophilic Phosphopeptides
Chia-Feng Tsai, Jeffrey S. Smith, Dylan S. Eiger, Kendall Martin, Tao Liu,
Richard D. Smith, Tujin Shi, Sudarshan Rajagopal, and Jon M. Jacobs
Abstract
Protein phosphorylation is a critical posttranslational modification (PTM), with cell signaling networks
being tightly regulated by protein phosphorylation. Despite recent technological advances in reversedphase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic
phosphopeptides that often have multiple phosphorylation sites. Herein, we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol
was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide
hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of
identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method,
we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor
(GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This
protocol is easy to adopt and implement and thus should have broad utility for effective RPLC-MS analysis
of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.
Key words Hydrophilic phosphopeptide, Phosphoproteomics, TMT labeling, Phosphopeptide
enrichment, Mass spectrometry
1 Introduction
Cell signaling is highly regulated by protein phosphorylation [1–
3]. Changes in phosphorylation stoichiometry are used as an indicator of signaling pathway activation (cellular functional states) [1–
3]. Aberrant protein phosphorylation is linked to human diseases
including cancer [2–4]. Mass spectrometry (MS)-based phosphoproteomics has emerged as a powerful tool for comprehensive, sitespecific, quantitative phosphoproteome profiling. Due to the low
stoichiometry of protein phosphorylation and low ionization efficiency of phosphopeptides, enrichment of phosphopeptides (e.g.,
Mo ´ nica Carrera and Jesu ´ s Mateos (eds.), Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 2259,
https://doi.org/10.1007/978-1-0716-1178-4_16, © Springer Science+Business Media, LLC, part of Springer Nature 2021
247