ReviewModificomics: Posttranslational modifications beyond protein phosphorylation and glycosylation
Introduction
Posttranslational modifications serve many different purposes in various cellular processes such as enzyme regulation, signal transduction, mediation of protein localization, interactions and stability. Genomic data can only partly be used for prediction of PTMs although specific software and databases are rapidly evolving (Blom et al., 2004, Chen et al., 2006, Lee et al., 2006, Xue et al., 2006). Therefore, proteomics is the method-of-choice for the analysis of modified proteins and peptides. The enormous versatility of the modifications that frequently alter the physicochemical properties of the respective proteins significantly is only one of the challenges of modification-oriented proteomics. Protein modifications are often transient, substoichiometric, time- and location-specific, site-specific and polymorphic (Sickmann et al., 2002). Thus, the analysis of posttranslational modifications is probably the most versatile and difficult, but also most frequently studied area of interest in proteomics research. This growing field of “modificomics” will yield many important insights into cellular networks but still face further challenges along with analytical and technical progress.
Although several hundreds of different modifications are known (Agris, 2004) protein phosphorylations and glycosylations and the respective analysis techniques are more often addressed by contemporary reviews (Harvey, 2005, Morandell et al., 2006, Morelle et al., 2006, Morelle and Michalski, 2005, Mukherji, 2005, Mumby and Brekken, 2005) than other modifications. Nevertheless, essential cellular functions are based on further PTMs such as protein lipidations, nitrosylations, sulfations or oxidative modifications. Thus, we will rather discuss techniques for the analysis of some of these posttranslational modifications that are equally important but have gained less attention during the last years.
A comprehensive analysis of different posttranslational modifications in parallel is usually not possible on a global scale. Therefore, the analysis should either be focussed on a single or very few distinct proteins or be directed towards a certain type of modification. Particularly the versatility, stoichiometry and dynamics of protein modifications raise the need for custom-made solutions for each issue to be addressed (Reinders et al., 2004). Therefore, all described methods represent rather general strategies that should be fitted to the respective matter than receipts to be followed step-by-step.
Section snippets
General considerations
Analysis strategies for posttranslational modifications solely depend on the intended purpose of the respective study. So the more you know about your sample and the clearer you can define your aim the bigger are your chances for a successful analysis.
The type of posttranslational modification to be analyzed will predefine most of the applicable sample preparation techniques, e.g. by its pH- and solvent-stability, influence on protein solubility or possible occurrence of artefacts. Furthermore,
Analysis of protein sulfation
Different types of protein sulfation (O-, S- and N-sulfation) are known (Huxtable, 1986) but sulfation of tyrosine residues occurring almost exclusively on secreted and membrane-spanning proteins is probably the best studied one (Hille et al., 1984, Hille et al., 1990, Hille and Huttner, 1990, Nemeth-Cawley et al., 2001). Furthermore, tyrosine sulfation is a more frequent modification than the much more thoroughly studied tyrosine phosphorylation (Monigatti et al., 2006) and is similarly
Analysis of deamidation
Conversion of Asn/Gln to Asp/Glu by deamidation is mostly not occurring during sample preparation and therefore not cause of artificial spots in 2D-electrophoresis. Particularly asparagines that are followed by glycine residues are susceptible to deamidation and are thought to serve regulatory purposes in the cell (Weintraub and Manson, 2004). Deamidation of asparagine and to a lesser extent glutamine side chains can either occur by direct hydrolysis of the amide group or by cyclic imide
Analysis of protein nitrosylation
Nitrosylation of proteins occurs upon modification with reactive nitrogen species such as peroxynitrite (ONOO−) or NOx and has been proposed as a marker for oxidative stress in both animal and plant biology (Kim et al., 2002, Schmidt and Walter, 1994, Shapiro, 2005). Proteins may be nitrosylated on cysteine residues leading to the formation of nitrosothiols (–SNO), on tyrosine residues generating 3-nitrotyrosine (–C6H4NO2OH) or on tryptophanes leading to different regioisomers of
Protein prenylation
Protein (iso-)prenylation is a lipid modification attaching farnesyl, dolichol or geranylgeranyl-moieties to cysteine residues close to the C-termini of proteins and often within a conserved motif, the so-called CAAX-box (Roskoski, 2003). These modifications are involved in recruitment of the modified proteins to membranes as well as facilitating protein interactions via prenyl-specific binding domains. For a long time the only method for detection of prenylated peptides was the introduction of
Analysis of protein oxidations
Reactive oxygen species (ROS) are generated upon oxidative stress introducing redox-modifications into proteins which are mostly studied by shifts in 2D-electrophoresis and subsequent mass spectrometry (Sheehan, 2006). Direct oxidation by the most important ROS, the hydroxyl radical OH, results in rather unspecific oxidation of proteins leading to protein inactivation and degradation via the ubiquitin-proteasome pathway (Poppek and Grune, 2006). Other ROS with lower oxidation potential
Concluding remarks
Modification-oriented proteomics is one of the fastest growing fields in proteomic research. While various techniques have been established for the analysis of phosphorylation and glycosylation suitable methods for the analysis of other, by no means less important protein modifications have only recently been developed or are still lacking. Thus, such modifications may come into the proteomic research focus even more in the near future bearing valuable information for the understanding of
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