Post-translational modification of proteins

Membrane and secretory proteins must be modified after translation to ensure proper cellular localization. 

Secretory and membrane-tai eted proteins are synthesized as larger precursors on endoplasmic reticulum (ER)—bound ribosomes, which inject the protein across or into the ER membrane as it is translated. 

Endoproteases cleave these proteins to activate them in the ER during sorting in the Golgi apparatus, during storage in secretory vesicles, or at the time of use when they arrive at their final destinations. 

Post-translational covalent modification of many proteins is important for their proper function and subcellular localization. 

Most secreted and membrane-embedded proteins are modified by addition of sugar structures (glycosylation) to the side chains of some amino acids. 

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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. 

After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking... 

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

Kivirikko, K.I., Myllyla, R. and Pihlajaniemi, T. (1992) Post Translational Modification of Proteins. CRC Press, Boca Raton, Florida, pp. 1-51. 

Staudinger ligation techniques also can be used to detect post-translational modification of proteins in vivo. Hang et al. (2007) developed a method to monitor fatty acid acylation of proteins using azido-fatty acids fed to cells. The two major types of fatty acid acylation,... 

The Golgi apparatus is a complex of folded membranes, which is involved in the post-translational modification of proteins. 

The Golgi apparatus (3) is a complex network, also enclosed, consisting of flattened membrane saccules ( cisterns ), which are stacked on top of each other in layers. Proteins mature here and are sorted and packed. A distinction is made between the ds, medial, and trans Golgi regions, as well as a trans Golgi network (tGN). The post-translational modification of proteins, which starts in the ER, continues in these sections. 

D. J. Graves, B. L. Martin J. H. Wang (1994) Co- and Post-Translational Modification of Proteins Chemical Principles and Biological Effects, pp. 348ff, Oxford University Press, Oxford. 

Krishna RG, Wold F (1993) Post-translational modification of proteins. Adv Enzymol Relat Areas Mol Biol 67 265-298... 

I27l Siegel, F. L., In Advances in Post-translational Modification of Proteins and Aging, Zappia, V. Galletti, P. Porta, R. Wold, F Eds. Plenum New York, (1988) p 341. 

Other post-translational modifications of proteins (e.g., phosphorylation) are extremely important mechanisms of regulation of enzyme activity. Very tittle work has been done on the effects of such modifications on enzyme stabilization. 

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. 

Graves, D. J., Martin, B. L., and Wang, J. H. (1994) Co-and post-translational modification of proteins, Oxford Univ. Press, New York... 

They can carry out post-translational modifications of proteins, but while the products resemble the products of human cells, they are not identical. 

Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd edition. W. H. Freeman, New York. Freeman, R. B., and Hawkins, H. C., Eds. (1980, 1985) The Enzymology of Post-translational Modifications of Proteins, Vols. 1 and 2. Academic Press, New York. 

In order to assign the disulfide bonds of these molecules fast atom bombardment mass spectrometry (FABMS) which has been used not only to confirm amino acid sequence data but also to elucidate post-translational modifications of proteins, such as disulfide bonds, has been employed. For this purpose a sample of native Er-2, containing four methionines, was subjected to CNBr cleavage and without further fractionation directly... 

This chapter describes some of the most important and well-studied forms of post-translational modifications of proteins, which have been associated with the production of glycoproteins as biopharmaceuticals from mammalian cell culture systems. Most attention is given to glycosylation because of its general importance for the activity of all these... 

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