Functional phosphoproteomic mass spectrometry-based approaches

Abstract

Mass Spectrometry (MS)-based phosphoproteomics tools are crucial for understanding the structure and dynamics of signaling networks. Approaches such as affinity purification followed by MS have also been used to elucidate relevant biological questions in health and disease.The study of proteomes and phosphoproteomes as linked systems, rather than research studies of individual proteins, are necessary to understand the functions of phosphorylated and un-phosphorylated proteins under spatial and temporal conditions. Phosphoproteome studies also facilitate drug target protein identification which may be clinically useful in the near future.Here, we provide an overview of general principles of signaling pathways versus phosphorylation. Likewise, we detail chemical phosphoproteomic tools, including pros and cons with examples where these methods have been applied. In addition, basic clues of electrospray ionization and collision induced dissociation fragmentation are detailed in a simple manner for successful phosphoproteomic clinical studies.

Figure 1

A possible proteomic work-flow for clinical studies. For identification, in-gel or in-solution proteins of interest are excised, washed and enzimatically digested to extract peptides for further analysis by MS. The mass to charge (m/z) ratio of the retained peptides can be measured by a mass spectrometer. Data-computer analysis is carried out which mainly includes: identification and classification of proteins, validation of identified protein, interpretation and classification of the protein lists, structural analysis of proteins, PTM analysis and Cellular composition.

Figure 2

Scheme of the three-tiered modules of the mitogen-activated protein kinases (MAPK) in eukaryotic cells. MAPK signaling cascades are organized hierarchically into three-tiered modules (a) (b) and (c). MAPKs are phosphorylated and activated by MAPK-kinases (MAPKKs), which in turn are phosphorylated and activated by MAPKK-kinases (MAPKKKs). MAPKKKs are in turn activated by interaction with the family of small GTPases and/or other protein kinases, connecting the MAPK module to cell surface receptors or external stimuli. Phosphoproteomics strategies allow identification of several phosphorylated proteins at a given time and space, thus it is possible to study the dynamic processes of signaling networks via phosphoproteomic MS-based tools (GPCR: G-protein coupled receptor).

Figure 3

Pros and cons of the most useful phosphoproteomic -MS based tools. Generally, immunoprecipitation of Tyrosine via antibodies, is more efficient than antibody immunoprecipitation of Serine and Threonine residues. On the other hand, affinity chromatography of IMAC and TiO₂, is more efficient for isolating phosphorylated residues of Serine and Threonine than Tyrosine. Moreover, TiO₂ has shown higher specificity when it is coupled to DHB than IMAC. In addition, IMAC has shown higher specificity when converting non-phosphorylated residues (acidic peptides) to methyl esters, but the complexity of spectra increases. Furthermore, SCX, SAX and calcium phosphate precipitation methods, are more efficient when coupled to IMAC and/or TiO₂ for clinical phosphoproteomic studies.

Figure 4

Illustration in a simple manner of the electrospray ionization process. The analyte solution is forced through the capillary, which has been supplied with high voltage. A Taylor cone is created due to the electric field between the capillary and the counter electrode, forming charged droplets of analyte ions and solvent. As these droplets travel towards the mass spectrometer the solvent evaporates creating analyte ions. When the solution that comprises the Taylor cone reaches the Rayleigh limit, at which point the Coulombic repulsion of the surface charge is equal to the surface tension of the solution, charged droplets are formed at the tip of the capillary. (Courtesy TE Tingholm Master Thesis 2005, PR Group, Odense Univ. Denmark).