Interpreting the protein language using proteomics


Post-translational modifications define the functional and structural plasticity of proteins in archaea, prokaryotes and eukaryotes. Multi-site protein modification modulates protein activity and macromolecular interactions and is involved in a range of fundamental molecular processes. Combining state-of-the-art technologies in molecular cell biology, protein mass spectrometry and bioinformatics, it is now feasible to discover and study the structural and functional roles of distinct protein post-translational modifications.

Key Points

  • Post-translational modifications (PTMs) affect protein conformation and interactions and can mediate the sequestration of proteins to cellular compartments and organelles.

  • The integration of molecular-cell-biology, protein-mass-spectrometry and bioinformatics technologies facilitates the study of protein PTMs.

  • Mass spectrometry identifies PTMs by looking for distinct mass increments or diagnostic PTM-specific signals that are generated by the tandem-mass-spectrometry analysis of peptides and proteins.

  • Modification-specific proteomics takes advantage of organelle purification and PTM-specific affinity-enrichment methods, multidimensional peptide-separation methods and tandem mass spectrometry to achieve high sensitivity and selectivity for the determination of PTMs.

  • The quantitative analysis of PTMs is possible. This can be achieved by using two-dimensional gel-based methods and imaging or by using liquid-chromatography–mass-spectrometry together with peptide-intensity-profiling or stable-isotope-labelling approaches.

  • Functional phosphoproteomics studies are delineating cellular signal-transduction cascades and revealing new regulatory mechanisms in microbes, plants and mammals.

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Figure 1: Cellular post-translational modifications.
Figure 2: Mechanism of action of post-translational modifications.


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I thank my colleagues in the Protein Research Group, as well as numerous colleagues in the world of proteomics, for many interesting and stimulating scientific discussions. I am grateful to M. B. Trelle for his assistance in preparing the figures. This manuscript is dedicated to the memory of my late father, Alex B. Jensen (1940–2005).

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Center for Biological Sequence Analysis Prediction Servers (Technical University of Denmark)

Human Protein Reference Database (HPRD)

HUPO Proteomics Standards Initiative

PhosphoELM (a database of S/T/Y phosphorylation sites)

Protein Research Group, University of Southern Denmark

The Association of Biomolecular Resource Facilities

UNIMOD (protein modifications for mass spectrometry)



A chromatin immunoprecipitation (ChIP) experiment that uses protein–DNA crosslinking, specific antibodies and cDNA microarrays ('chips') for large-scale studies of DNA-binding proteins, including the analysis of post-translational modifications.


Nanolitre-flow chromatographic systems for protein and peptide separations prior to mass-spectrometry analysis.

Electrospray ionization

A 'soft' ionization method for mass spectrometry that generates gas-phase ions from peptide and protein solutions through the vapourization of liquid in an electric field.

Matrix-assisted laser desorption/ionization

A 'soft' ionization technique for mass spectrometry that produces gas-phase ions through the pulsed, ultraviolet laser irradiation of crystalline deposits of peptides and proteins.

TiO2 columns

Titanium dioxide interacts with phosphate groups by a chelation mechanism. These columns are therefore useful for the selective and specific enrichment of phosphopeptides.

β-elimination reaction

The alkaline-induced elimination of phosphoric acid from phosphoserine and phosphothreonine residues or of O-linked sugars from O-glycosylated residues in peptides.

Michael addition reaction

The addition of a functional chemical moiety to a residue that was post-translationally modified but that has undergone a β-elimination reaction. Affinity tags or mass tags can be added to allow the specific recovery or detection of post-translationally modified peptides.

Phosphoramidate chemistry

A method for the selective capture of converted post-translationally modified peptides, with the aim of identifying the modified protein and the site of the modification.

Chromatographic stationary phases

The column resins that are used for the chromatographic separation of molecules — for example, a reversed-phase resin or a strong-cation-exchange resin.

Cell-signalling cassettes

Multiprotein modules that are involved in signal-transduction processes and that rapidly propagate stimuli from cell-surface receptors to the cytosol or the nucleus.

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