This Title All WIREs
How to cite this WIREs title:
WIREs Dev Biol
Impact Factor: 5.814

Mass spectrometry‐driven phosphoproteomics: patterning the systems biology mosaic

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Protein phosphorylation is the best‐studied posttranslational modification and plays a role in virtually every biological process. Phosphoproteomics is the analysis of protein phosphorylation on a proteome‐wide scale, and mainly uses the same instrumentation and analogous strategies as conventional mass spectrometry (MS)‐based proteomics. Measurements can be performed either in a discovery‐type, also known as shotgun mode, or in a targeted manner which monitors a set of a priori known phosphopeptides, such as members of a signal transduction pathway, across biological samples. Here, we delineate the different experimental levels at which measures can be taken to optimize the scope, reliability, and information content of phosphoproteomic analyses. Various chromatographic and chemical protocols exist to physically enrich phosphopeptides from proteolytic digests of biological samples. Subsequent mass spectrometric analysis revolves around peptide ion fragmentation to generate sequence information and identify the backbone sequence of phosphopeptides as well as the phosphate group attachment site(s), and different modes of fragmentation like collision‐induced dissociation (CID), electron transfer dissociation (ETD), and higher energy collisional dissociation (HCD) have been established for phosphopeptide analysis. Computational tools are important for the identification and quantification of phosphopeptides and mapping of phosphorylation sites, the deposition of large‐scale phosphoproteome datasets in public databases, and the extraction of biologically meaningful information by data mining, integration with other data types, and descriptive or predictive modeling. Finally, we discuss how orthogonal experimental approaches can be employed to validate newly identified phosphorylation sites on a biochemical, mechanistic, and physiological level. WIREs Dev Biol 2014, 3:83–112. doi: 10.1002/wdev.121 This article is categorized under: Technologies > Analysis of Proteins

This WIREs title offers downloadable PowerPoint presentations of figures for non-profit, educational use, provided the content is not modified and full credit is given to the author and publication.

Download a PowerPoint presentation of all images

Systems biology (a) and proteomic (b) research are interdisciplinary fields. Fruitful research in these areas depends on strong intersections and dialog between experts in biology, technology, and computation.
[ Normal View | Magnified View ]
Different levels of validation for newly discovered phosphorylation sites. We divide the various types of validation experiments into three categories of varying biological information content, namely characterization of the phosphorylation events itself, investigation of the mechanistic relevance of the modification for the protein on which it occurs, and assessment of in vivo relevance by genetic means and structure–function analyses.
[ Normal View | Magnified View ]
Phosphoproteomics in the framework of systems biology. Phosphoproteome profiles can be either analyzed as stand‐alone datasets, or combined with other experimental data (yellow ovals) to generate further biological knowledge. In addition, experimental datasets of different sources which are stored in public databases or retrieved from literature can be integrated with the acquired phosphoproteome profiles in various ways. The generation of descriptive or predictive models of processes involving phosphorylation events depend on high‐quality experimental data describing different cellular parameters.
[ Normal View | Magnified View ]
Quantitation strategies in phosphoproteomics. The triple color coding represents the three distinct analytical stages of LC (blue), MS1 (green), and MS2 (orange) during LC‐MS/MS. The different quantitation schemes are colored according to which kind of information they exploit for quantitation. The yellow boxes on the left indicate categories such as label‐free methods or stable isotope labeling that are commonly used for the different quantitation strategies. Note that in contrast to spectral counting, SIL techniques can also be combined with strategies that are also used for label‐free quantification. For example, chemical tags with a mass shift such as ICAT or ICPL, or sample preparation methods employing metabolic labeling or synthetic heavy peptides can be used in conjunction with MS1‐based quantification or SRM, and isobaric tags like iTRAQ or TMT can be combined with SRM.
[ Normal View | Magnified View ]
Mass spectrometry of phosphopeptides. The different elements of mass spectrometric analysis of (phospho‐) peptides are shown. The central element is the fragmentation of peptides which generates sequence information and is therefore instrumental in identifying peptides in a biological sample. Different fragmentation strategies used in phosphoproteomics are displayed, as are the diverse acquisition schemes that have already been introduced in Figure .
[ Normal View | Magnified View ]
Experimental strategies to extend phosphoproteome coverage and introduce stable isotope‐labeled reference molecules. The blue boxes on the left display the typical stages in a phosphoproteomic experiment. The boxes in the central part of the figure show experimental processes either during (mauve) or between (yellow) the stages, which can impact the success (in terms of phosphoproteome coverage and quality of quantitation) of the experiment. The orange boxes on the right illustrate at which stages stable isotope labels can be introduced. Introduction of isotope labels at an early experimental stage reduces individual sample handling errors because the samples are pooled earlier, and therefore usually leads to a more accurate quantification.
[ Normal View | Magnified View ]
Approaches to the analysis of protein phosphorylation. Experimental categories employing radioactivity, antibody‐based detection, and mass spectrometry are shown. Refer to the text for more details.
[ Normal View | Magnified View ]
Signaling pathways and kinase–substrate relationships in biological systems. Three schematic pathway examples of increasing complexity are shown. (a) Bacterial two‐component signaling consisting of histidine kinases (HK) and response regulators (RR) constitutes one of the simplest signaling systems, while eukaryotic cells feature networks such as the relatively simple JAK‐STAT pathway (b) and the more complex insulin‐TOR network (c). Only the core constituents of the pathways are shown for the sake of clarity. Interconnected cascades of direct kinase–substrate relationship form the functional backbone of these networks. Another important concept in pathway architecture is the role of phosphospecific protein binding modules like SH2 domains and 14‐3‐3 proteins in the dynamic regulation of phosphorylation dependent protein complexes.
[ Normal View | Magnified View ]

Browse by Topic

Technologies > Analysis of Proteins