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Proteomics has rapidly evolved from the detection and cataloguing of the protein complement of either a cell, or sub-cellular organelle, or complex through the quantitative characterisation of proteins and their interactions to now encompass the comprehensive, large-scale quantitative study of protein dynamics. This includes measuring changes in the complex pattern of post-translational modifications (PTMs) in space and time. Here, we will refer to this new state of the art as “Next Generation” proteomics, reflecting the current depth and detail of analysis that is possible. We anticipate that the application of these new proteomics methods will revolutionise our understanding of cellular function. We will review methods for the large-scale, spatial and temporal quantitative analysis of some of the most commonly studied PTMs in mammalian cell lines, including phosphorylation, acetylation, ubiquitinylation and hydroxylation. We will focus our discussion on the use of mass spectrometry (MS) combined with stable-isotopic labelling for the quantification of proteins and PTMs, because this has been used so extensively in recent cell biology and cell signalling studies and we will contrast this with label-free methods. We present here an example of an optimised workflow, from sample preparation, through sub-cellular fractionation and liquid chromatography, to data acquisition using high resolution and high mass accuracy Fourier transform MS. An integral part of this “Next Generation” workflow includes methods for the efficient storage, analysis, visualisation and sharing of the resulting data. Methods will be described for maximising the protein properties that can be measured in a single experiment by combining appropriate sample preparation, data acquisition and analytical methods. Examples will be shown that illustrate the detection of PTMs and show how these data can be correlated with other measured protein properties, including sub-cellular localisation, in system-wide studies covering a large fraction of the expressed cell proteome.

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