Review
Modificomics: Posttranslational modifications beyond protein phosphorylation and glycosylation

https://doi.org/10.1016/j.bioeng.2007.03.002Get rights and content

Abstract

Posttranslational modifications of proteins possess key functions in the regulation of various cellular processes. While they facilitate fast, location-specific and transient reactions to changing conditions in the first place they enhance the already high complexity of a cellular proteome by orders of magnitude. Furthermore, they can utterly alter the properties of the modified protein, thus making a timely analysis even more difficult. While several standardized methods for the analysis of protein phosphorylation and glycosylation have been established most other modifications require tailor-made solutions for a comprehensive analysis. Therefore, we will provide guidelines for the analysis of some important posttranslational modifications that are underrepresented in contemporary literature.

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 radical dotOH, 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

References (79)

  • K. Ikeda et al.

    Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells

    Nitric Oxide

    (2007)
  • R.L. Levine et al.

    Determination of carbonyl content in oxidatively modified proteins

    Methods Enzymol.

    (1990)
  • I. Navarro-Lerida et al.

    Palmitoylation of inducible nitric-oxide synthase at Cys-3 is required for proper intracellular traffic and nitric oxide synthesis

    J. Biol. Chem.

    (2004)
  • P. Onnerfjord et al.

    Identification of tyrosine sulfation in extracellular leucine-rich repeat proteins using mass spectrometry

    J. Biol. Chem.

    (2004)
  • J.L. Reubsaet et al.

    Analytical techniques used to study the degradation of proteins and peptides: chemical instability

    J. Pharm. Biomed. Anal.

    (1998)
  • R. Roskoski

    Protein prenylation: a pivotal posttranslational process

    Biochem. Biophys. Res. Commun.

    (2003)
  • A.F. Roth et al.

    Global analysis of protein palmitoylation in yeast

    Cell

    (2006)
  • A.F. Roth et al.

    Proteomic identification of palmitoylated proteins

    Methods

    (2006)
  • H.H. Schmidt et al.

    NO at work

    Cell

    (1994)
  • A.D. Shapiro

    Nitric oxide signaling in plants

    Vitam. Horm.

    (2005)
  • D. Sheehan

    Detection of redox-based modification in two-dimensional electrophoresis proteomic separations

    Biochem. Biophys. Res. Commun.

    (2006)
  • E.R. Stadtman et al.

    Methionine oxidation and aging

    Biochim. Biophys. Acta

    (2005)
  • S.W. Taylor et al.

    Oxidative post-translational modification of tryptophan residues in cardiac mitochondrial proteins

    J. Biol. Chem.

    (2003)
  • S. Toyokuni et al.

    The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct

    FEBS Lett.

    (1995)
  • K. Uchida

    Role of reactive aldehyde in cardiovascular diseases

    Free Radic. Biol. Med.

    (2000)
  • S.J. Weintraub et al.

    Asparagine deamidation: a regulatory hourglass

    Mech. Ageing Dev.

    (2004)
  • S. Yamada et al.

    Immunochemical detection of a lipofuscin-like fluorophore derived from malondialdehyde and lysine

    J. Lipid Res.

    (2001)
  • S. Yamada et al.

    Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n  3 polyunsaturated fatty acids

    J. Lipid Res.

    (2004)
  • F. Yamakura et al.

    Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species

    Nitric Oxide

    (2006)
  • K. Zhu et al.

    Use of two-dimensional liquid fractionation for separation of proteins from cell lysates without the presence of methionine oxidation

    J. Chromatogr. A

    (2004)
  • P.F. Agris

    Decoding the genome: a modified view

    Nucleic Acids Res.

    (2004)
  • A.C. Antony et al.

    Statistical prediction of the locus of endoproteolytic cleavage of the nascent polypeptide in glycosylphosphatidylinositol-anchored proteins

    Biochem. J.

    (1994)
  • K.S. Aulak et al.

    Proteomic method for identification of tyrosine-nitrated proteins

    Methods Mol. Biol.

    (2004)
  • N. Blom et al.

    Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence

    Proteomics

    (2004)
  • G.H. Borner et al.

    Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

    Plant Physiol.

    (2005)
  • H. Chen et al.

    MeMo: a web tool for prediction of protein methylation modifications

    Nucleic Acids Res.

    (2006)
  • J.M. Cordy et al.

    Exclusively targeting beta-secretase to lipid rafts by GPI-anchor addition up-regulates beta-site processing of the amyloid precursor protein

    Proc. Natl. Acad. Sci. U.S.A.

    (2003)
  • E.M. Danielsen

    Tyrosine sulfation, a post-translational modification of microvillar enzymes in the small intestinal enterocyte

    EMBO J.

    (1987)
  • R.C. Drisdel et al.

    Labeling and quantifying sites of protein palmitoylation

    Biotechniques

    (2004)
  • Cited by (71)

    • Protein post-translational modifications – A challenge for bioelectrochemistry

      2019, TrAC - Trends in Analytical Chemistry
      Citation Excerpt :

      Over the past two decades, protein kinases, i.e. the enzymes responsible for protein phosphorylation, have become one of the most important drug targets, especially for anti-cancer drugs development [17,18]. Recent advances in research methods allowed to obtain deeper insights into the mechanisms of PTMs formation, their effects on the structure and function of proteins and their disease relevance [19,25,26]. One should note that genomics per se is unable to predict the particular appearance of proteins such as their PTMs.

    • Electrochemical methods for detection of post-translational modifications of proteins

      2014, Biosensors and Bioelectronics
      Citation Excerpt :

      Post-translational 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 (Jia et al., 2013; Reinders and Sickmann, 2007). Modern proteomics is the main method for the analysis of modified proteins and peptides, and the analysis of post-translational modifications is probably the most difficult area in proteomics.

    • An overview of proteomics approaches applied to biopharmaceuticals and cyclotides research

      2013, Journal of Proteomics
      Citation Excerpt :

      PTMs are basically divided into two main groups: covalent cleavage of side chains and covalent attachment of chemical groups [18]. About 40 % of proteins employed in therapeutics are N-glycosylated [24,52] and, in plants, the process is quite well characterized under proteomics and molecular biology approaches [16,53], being similar to other eukaryotic organisms in terms of frequency of glycosylation [54]. Even thought PTMs are known to be evolutionarily conserved, there are plant specific PTMs that should be taken into consideration when proteins are to be expressed in plant systems through genetic engineering [53].

    View all citing articles on Scopus
    View full text