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Review
. 2017 Feb 23;4(Pt 2):119-130.
doi: 10.1107/S2052252516020546. eCollection 2017 Mar 1.

Advances in Mass Spectrometry Based Strategies to Study Receptor Tyrosine Kinases

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Free PMC article
Review

Advances in Mass Spectrometry Based Strategies to Study Receptor Tyrosine Kinases

Simon Vyse et al. IUCrJ. .
Free PMC article

Abstract

Receptor tyrosine kinases (RTKs) are key transmembrane environmental sensors that are capable of transmitting extracellular information into phenotypic responses, including cell proliferation, survival and metabolism. Advances in mass spectrometry (MS)-based phosphoproteomics have been instrumental in providing the foundations of much of our current understanding of RTK signalling networks and activation dynamics. Furthermore, new insights relating to the deregulation of RTKs in disease, for instance receptor co-activation and kinome reprogramming, have largely been identified using phosphoproteomic-based strategies. This review outlines the current approaches employed in phosphoproteomic workflows, including phosphopeptide enrichment and MS data-acquisition methods. Here, recent advances in the application of MS-based phosphoproteomics to bridge critical gaps in our knowledge of RTK signalling are focused on. The current limitations of the technology are discussed and emerging areas such as computational modelling, high-throughput phospho-proteomic workflows and next-generation single-cell approaches to further our understanding in new areas of RTK biology are highlighted.

Keywords: cancer; mass spectrometry; phosphoproteomics; receptor tyrosine kinase; signal transduction.

Figures

Figure 1
Figure 1
Overview of mass spectrometry-based phosphoproteomic workflows. Depending on the experimental design, there are a number of different strategies which can be chosen to enrich specific compartments of the phosphoproteome. This is commonly followed by fractionation to reduce complexity and increase coverage of complex cell lysates. Finally, the method of data acquisition will be influenced by the specific biological questions of interest when interrogating proteomic data.
Figure 2
Figure 2
Advantages and disadvantages of enrichment and acquisition methods in phosphoproteomic workflows. A comparison of the benefits and drawbacks of chemical, immunoaffinity and small-molecule-based phosphoproteome enrichment and data-dependent (DDA), data-independent (DIA) and selective reactive monitoring (SRM) acquisition methods, which must be considered when designing phosphoproteomic experiments.
Figure 3
Figure 3
Schematic comparison of mass-spectrometric data-acquisition methodologies. (a) DDA: precursors identified in the first MS1 stage are selected for MS2 fragmentation on the basis of abundance. Software matches the masses to the database (in silico ‘trypsinized’ proteins). This is the standard discovery mode allowing the identification of novel proteins and phosphorylation sites. (b) SRM: precursors chosen on basis of prior discovery experiments in the MS1 stage; following fragmentation, signature MS2 peaks are also selected. The integration of these transitions can be used for quantitation. (c) DIA: no precursor selection in the MS1 stage; instead, all ions in wide overlapping mass windows (typically 25 mass units) over the whole mass range (from 400 to 1200 m/z) are fragmented. Using spectral libraries obtained in DDA experiments, MS2 spectra corresponding to specific peptides can be extracted.
Figure 4
Figure 4
Advances in understanding RTK biology using mass spectrometry-based phosphoproteomic studies. A timeline of key studies which illustrate the development of MS-based phosphoproteomics and their application in advancing our knowledge of RTK biology. The timeline depicts the pioneering phosphoproteomic studies performed a decade ago in addition to highlighting novel and innovative research from the last five years.

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