Molecular post-translational modification

While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

Post-translational modifications to proteins are biochemical in origin and alter the measured molecular mass relative to that calculated for an untranslated sequence. 

Protein macromolecules present in biological fluids are almost invariably heterogeneous in their characteristics. They may be products of more than one gene in the population (allotypes in the case of proteins isoenzymes in the case of enzymes), or a single individual (isotypes of proteins, allelozymes of enzymes), or be subject to post-translational modification. The result of this inherent molecular heterogeneity is that different forms of the same protein may behave differently with respect to... 

Several steps were needed to determine the structure of the core particle to higher resolution (Fig. Id). The X-ray phases of the low-resolution models were insufficient to extend the structure to higher resolution, since the resolution of the early models of the NCP was severely limited by disorder in the crystals. The disorder was presumed to derive from both the random sequences of the DNA and from heterogeneity of the histone proteins caused by variability in post-translational modification of the native proteins. One strategy for developing an atomic position model of the NCP was to develop a high-resolution structure of the histone core. This structure could then be used with molecular replacement techniques to determine the histone core within the NCP and subsequently identify the DNA in difference Fourier electron density maps. 

Williams, K.L. and Hochstrasser, D.F. (1999) High-throughput mass spectrometric discovery of protein post-translational modifications. Journal of Molecular Biology, 289, 645-657. 

Immunoprecipitation followed by SDS-PAGE and immunoblotting, is routinely used in a variety of applications to determine the molecular weights of protein antigens, to study protein-protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to... 

With respect to sample preparation, it is necessary to develop effective and fast procedures involving only a few steps in order to avoid contamination, reduce analysis time and to improve the quality of analytical work. Microsampling and the use of smaller sample sizes is required and also the further development of analytical techniques. In particular, there is a need for the development of online and/or hyphenated techniques in ICP-MS. Microsampling combined with the separation of small amounts of analytes will be relevant for several chromatographic techniques (such as the development of micro- and nano-HPLC). There is a demand for further development of the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spectrometric technique such as MALDI- or ESI-MS for molecular identification and quantification of protein phosphorylation as well as of metal concentrations, this also enables the study of post-translational modifications of proteins, e.g. phosphorylation. 

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