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WIREs Dev Biol
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Regulation of developmental processes: insights from mass spectrometry‐based proteomics

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Abstract Mass spectrometry (MS)‐based proteomics has become an indispensable tool for protein identification and quantification. In this paper, common MS workflows are described, with an emphasis on applications of MS‐based proteomics in developmental biology. Progress has been made in the analysis of proteome changes during tissue differentiation and in various genetic perturbations. MS‐based proteomics has been particularly useful for identifying novel protein interactions by affinity purification‐mass spectrometry (AP‐MS), many of which have been subsequently functionally validated and led to the discovery of previously unknown modes of developmental regulation. Quantitative proteomics approaches can be used to study posttranslational modifications (PTMs) of proteins such as phosphorylation, to reveal the dynamics of intracellular signal transduction. Integrative approaches combine quantitative MS‐based proteomics with other high‐throughput methods, with the promise of a systems level understanding of developmental regulation. WIREs Dev Biol 2013, 2:723–734. doi: 10.1002/wdev.102 This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes Technologies > Analysis of Proteins

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Common mass spectrometry‐based proteomics workflows. (a) Shotgun proteomics. The goal of shotgun proteomics experiments is to discover and identify as many proteins and their posttranslational modifications (PTMs) as possible. An example fragmentation process in the collision cell is shown for a random peptide; this process is repeated for all detected peptides. (b) Targeted proteomics: selected reaction monitoring (SRM). The aim of targeted proteomics studies is to quantify a defined, limited set of proteins with high reproducibility and sensitivity. A single precursor peptide and its single fragment chosen for SRM are highlighted in red throughout the procedure. (c) Quantitative proteomics. The goal is to quantify differences in protein abundance between two or more samples, which is achieved by differential labeling of proteins or peptides with stable isotopes. This approach can be used both with shotgun and targeted proteomics methods. (d) Analysis of PTMs. Identification of PTMs is possible due to a mass shift that is characteristic for a particular modification. In the example shown, phosphorylation shifts the mass of a peptide by 80 Da. Abbreviations: LC, liquid chromatography; ESI, electrospray ionization. See text for more details.

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Affinity purification‐mass spectrometry (AP‐MS) workflow. A protein of interest is tagged with an affinity tag and expressed in cultured cells or in vivo. The bait protein and associated subunits are purified using one or more affinity steps, followed by LC‐MS/MS identification of components. Novel associations are validated in functional studies.

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