Review
Targeted mass spectrometry: An emerging powerful approach to unblock the bottleneck in phosphoproteomics

https://doi.org/10.1016/j.jchromb.2017.04.026Get rights and content

Highlights

  • ā€¢

    Shotgun phosphoproteomic studies identify thousands of phosphosites.

  • ā€¢

    Traditional methods are insufficient to validate all phosphosites.

  • ā€¢

    Despite challenging targeted MS is the method of choice for phosphosite validation.

  • ā€¢

    Site-specific phosphorylation events are emerging as potential disease biomarkers.

  • ā€¢

    Targeted phosphoproteomic approaches could be relevant and useful in clinical applications.

Abstract

Following the rapid expansion of the proteomics field, the investigation of post translational modifications (PTM) has become extremely popular changing our perspective of how proteins constantly fine tune cellular functions. Reversible protein phosphorylation plays a pivotal role in virtually all biological processes in the cell and it is one the most characterized PTM up to date. During the last decade, the development of phosphoprotein/phosphopeptide enrichment strategies and mass spectrometry (MS) technology has revolutionized the field of phosphoproteomics discovering thousands of new site-specific phosphorylations and unveiling unprecedented evidence about their modulation under distinct cellular conditions. The field has expanded so rapidly that the use of traditional methods to validate and characterize the biological role of the phosphosites is not feasible any longer. Targeted MS holds great promise for becoming the method of choice to study with high precision and sensitivity already known site-specific phosphorylation events. This review summarizes the contribution of large-scale unbiased MS analyses and highlights the need of targeted MS-based approaches for follow-up investigation. Additionally, the article illustrates the biological relevance of protein phosphorylation by providing examples of disease-related phosphorylation events and emphasizes the benefits of applying targeted MS in clinics for disease diagnosis, prognosis and drug-response evaluation.

Introduction

Protein phosphorylation is one of the most widespread post-translational modifications (PTM) that plays an essential role in cellular physiology. Site-specific phosphorylations affect numerous critical aspects including protein activity, sub-cellular localization, conformation, stability and binding capacity to other molecules [1], [2], [3], [4], [5], [6]. Environmental cues such as mechanical stress, cell-cell interactions, growth factors or cytokines, trigger the activation of signaling networks that are mainly orchestrated by protein phosphorylation events [7], [8], [9]. It is estimated that at least one third of the proteins within a cell becomes phosphorylated at some point [10], consequently, it is assumed that virtually all cellular processes are directly or indirectly modulated by this PTM. Given the relevance of protein phosphorylation in modulating the physiology of the cell, it is not surprising that aberrant phosphorylation has been linked to a vast number of pathologies [11], [12]. Hence, phosphoproteins have emerged as potential biomarkers and major efforts are being devoted to developing robust and sensitive strategies for the detection of site-specific phosphorylations within complex biological samples.

The phosphorylation status of a protein is tightly modulated by the coordinated action of kinases and phosphatases that are responsible for attaching or removing the phosphate moiety, respectively. Usually, subtle changes in the phosphorylation levels are sufficient to alter the behavior of a protein. Nevertheless, in certain cellular processes, such as mitosis, half of the phosphosites detected have been demonstrated to present occupancy of at least 75% [13]. In addition to the overall substoichiometric nature of site-specific modifications, protein phosphorylation events are temporally modulated. Therefore, whereas a specific residue could be phosphorylated right after a specific stimulus, it might become unmodified after a while and vice versa [14], [15]. Precisely, among the three phosphorylable amino acids (Ser, Thr and Tyr) tyrosines are the less abundant but frequently regulated residues [14], [16]. Additionally, the regulation of phosphoproteins can be restricted to certain cellular compartments [13], [17], [18]. All these challenges are further complicated when multiple phosphorylation sites are present on the same phosphopeptide. In this scenario, it is crucial to determine the specific residue or residues that are modified in order to characterize the biological significance of such phosphorylation.

Traditionally, protein phosphorylation has been investigated using various biochemical approaches that rely on the use of radioactivity (32P), phospho-specific dyes or in vitro kinase assays [19], [20], [21]. Although the contribution of these types of studies is invaluable, they do not provide information about the precise site or sites that are modified within the phosphoprotein. Genetic approaches such as site-directed mutagenesis have greatly expanded our knowledge about biological role of the site specific phosphorylations occurring in proteins [22], [23], [24]. The increasing number of commercially available phosphosite-specific antibodies also allows detecting relatively easily whether a protein is phosphorylated or not on a certain residue that has been previously described. Nevertheless, phospho-site recognition is often hampered due to the antibodiesā€™ specificity and sensitivity issues. At present, mass spectrometry (MS) offers the most suitable approach for mapping phosphorylation sites and discovering novel modifications. The gain of mass resulting from the presence of the phosphate moiety is used to assign the specific residue subjected to modification. Indeed, breakthrough developments in the field of MS-based phosphoproteomics allow the detection and quantification of thousands of phosphosites simultaneously, which has greatly expanded our knowledge of site-specific protein modification as never before [25], [26]. The amount of novel information regarding phosphosites increases so rapidly that none of the classical techniques mentioned above can cope with the speed. Yet again, another MS-based strategy called targeted phosphoproteomics has emerged as a powerful resource to systematically monitor site-specific protein phosphorylation with high precision and sensitivity [27].

Here we review the contribution of MS-based phosphoproteomics to globally decipher phosphorylation in the cell, reviewing the ground covered from shotgun proteomics to targeted proteomics; we discuss the advantages and challenges of the targeted methodology and its promising applications in phosphosite quantification-based diagnosis of diseases.Ģ

Section snippets

Shotgun phosphoproteomics: an explosion on the identification of site-specific phosphorylation

The field of phosphoprotemics experienced a great revolution upon the development of strategies to enrich phosphopeptides and phosphoproteins prior MS analysis, thus overcoming the challenging substoichiometric nature of this transient PTM.

One of the most common phospho enrichment techniques is the immobilized metal affinity chromatography (IMAC) which uses metal ions such as Fe3+, Ga3+, Ti4+ or Zr4+ to bind the negatively charged phosphopeptides. Although the potential of IMAC to isolate

From shotgun analyses to targeted MS

Shotgun or discovery proteomics, based on Data Dependent Acquisition (DDA) mode, is a very powerful technique to explore complex biological samples and it is the most widely used MS approach for protein and PTM identification and quantification so far [61]. It requires no previous knowledge of the composition of the sample and it is the method of choice in early discovery phase studies. By contrast, targeted MS aims to detect and measure the abundance of peptides or proteins of interest without

Targeted phosphoproteomics

Mass spectrometry has become routine for PTM studies, in particular for phosphorylation analysis; among the different MS strategies available to date, SID-SRM is the most specific, sensitive and accurate method to quantify site-specific protein phosphorylations. However, targeted quantification of specific phosphopeptides presents still considerable analytical challenges. Essentially, the targeted phosphopeptide analysis follow the same acquisition scheme as for the analysis of unmodified

Conclusions and perspectives

Targeted approaches as tools for validating shotgun MS data generated in basic research are more and more in demand and the method of choice especially when commercial antibodies are unavailable or unsuitable due to lack of sensitivity or specificity. In addition, the samples collected in clinical settings are still challenging in terms of limited starting amount, heterogenecity and variability. Strategies focused on increased sensitivity of the phosphoproteomics analyses have been recently

Acknowledgments

This project has been funded by a grant from the Lundbeck Foundation, from the Danish National Research Council Medical Sciences and by the University of the Basque Country. Proteomics Core Facility-SGIKER is member of ProteoRed-ISCIII and supported by UPV/EHU, MINECO, GV/EJ, ERDF and ESF.

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