Protein post-translational modification analysis

Protein and post-translational modifications

Protein post-translational modifications (PTMs) are part of a complex regulatory network that controls physiological and pathological cellular processes. In this post, new user-friendly assays are introduced to help Life Scientists gain a deeper understanding of these mechanisms involved in protein biology.

Ubiquitin structure

Fig 1: Structure of Ubiquitin

Upon translation, proteins are trafficked to their proper subcellular location so that they can perform their physiological functions. One mechanism that mediates correct protein localisation and function are post-translational modifications (PTMs).

PTMs include protease cleavage, protein folding, and the attachment of molecules such as biochemical functional groups (e.g., acetate or phosphate) or small proteins (e.g., Ubiquitin [see Fig 1], or small Ubiquitin-like Modifier = SUMO, phosphorylation…). In this way, PTMs provide cells with a second level of regulation of protein function, signaling, and fate.

PTM-mediated regulation involves alteration of protein binding motifs, thereby affecting the binding with other proteins, nucleic acids, and various small molecules. Enzyme-mediated reversible PTMs offer an especially powerful modulatory tool that enable proteins to perform multiple tasks systematically while the protein remains under precise regulatory control.

In short, PTMs extend the function of proteins and influence almost all aspects of normal cell biology and pathogenesis.

PTMs include, but are not limited to, phosphorylations, sumoylations, ubiquitinations, glycosylations, acetylations… All these modifications have a biological meaning, changing the way a protein acts in the cell… and in the organism.

It has been shown that a number of proteins are modified by more than one unique PTM and that these modifications can cross-talk and build up complex regulatory networks.

SUMOylated tau protein for instance has been observed in late stage Alzheimer’s Disease (AD), and this correlated with reduced clearance of AD-tau via the ubiquitin proteasome system. The following cascade of PTM events has been reported:

  • A- SUMOylation on tau induces hyperphosphorylation,
  • B- Tau hyperphosphorylation enhances SUMOylation, and finally,
  • C- SUMOylation inhibits tau ubiquitination and degradation of AD-tau.

In parallel, Rolipram, a PDE inhibitor, has been described a to slow tau accumulation in AD models via the modulation of the proteasome

Schematic showing Signal-SeekerAssay Flow tebu-bio Cytoskeleton

Fig 2:  Signal-Seeker assay worlkflow to evaluate protein PTMs

The analysis of key regulatory molecular mechanisms involved in protein PTMs require a huge investment of time and effort.

To make such an investigation more user-friendly, Cytoskeleton Inc. has developed a new line of assay kits: the Signal-Seeker kits.

Protein post-translational modification analysis now made easier

Signal-Seeker assays enable a biologically relevant, hence simple, assessment of the key PTMs (ex. tyrosine phosphorylationubiquitination, acetylation and SUMOylation) of a protein or a pathway of interest.

The methodology is based on affinity beads to pull-out and enrich post-transcriptionally modified proteins from any given cell or tissue lysate (as seen on Fig 2).

The enriched protein population is then analyzed by standard Western Blot procedures; the protein of interest being detected by using a target-specific primary antibody (Fig 3).

Typical results obtained with the Signal Seeker assay to study Tyrosine phosphorylation results

Fig 3: Rac1 Tyrosyl phosphorylation and activation  detected with phosphotyr Signal Seeker Kit (Western blot with anti-Rac1 (R) and anti-Tubulin (T) antibodies on HeLa cells)

By using single signal-seeker kits or combining them you can:

  • Discover and publish novel regulatory mechanisms
  • Detect highly transient regulation of protein modifications
  • Confirm data generated from transfection or proteom approaches
  • Build up a temporal protein regulation profile (by using different kits)
  • Investigate the role of known protein modifications in your system
  • Obtain data for endogenous proteins
  • Discover novel biomarkers

Looking for Phosphorylation or Ubiquitination protein post-translational modifications?

The first two kits which have been released can be used to measure tyrosine phosphorylation and ubiquitination of your protein of interest. Kits related to Acetylation and SUMOylation will be available soon.

What about you? Which PTM are you interested in? Feel free to leave your comment below.

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Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.