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.

Protein phosphorylation and photorespiration

“…; Lopez et al . ). Low abundance phospho‐peptides are often poorly purified or in quantities too low to be detected with mass spectroscopy.…”

“…In nearly all cases, extracted plant proteins are trypsin-digested (and after this stage they can be labelled with stable isotopes to carry out quantitative analyses) and then these peptides are subjected to one or more phospho-peptide enrichment chromatography steps, such as strong cation exchange (SCX), immobilised metal affinity purification (IMAC, often with Fe 3+ ) or titanium oxide (TiO 2 ) chromatography. However, each enrichment option has certain limitations (see Kosako & Nagano 2011;Imamura et al 2012;Lopez et al 2012). Low abundance phospho-peptides are often poorly purified or in quantities too low to be detected with mass spectroscopy.…”

Study of phosphorylation events for cancer diagnoses and treatment

“…Nevertheless, the MS3 mode requires that the phosphorylation on ser and thr residues are labile and conventional fragmentation via CID commonly results in the partial NL of H 3 PO 4 , (98 m/z) in MS2 mode. This is due to the gas phase β‐elimination of the phosphor‐ester bond and thus, dehydroalanine (ser ~69 Da) and dehydro‐2‐aminobutyric acid (thr ~83 Da) are generated [36–48].…”

The significant others: Global search for direct kinase substrates using chemical approaches

“…In such instances, targeting its pathological substrates provides a superior option for developing effective therapeutics and for combating collateral toxicity . Interestingly, recent advancements in mass spectroscopy have enabled rapid profiling of the phosphoproteome with accurate determination of phosphorylation sites . However, it remains a challenge to dissect the contribution of a single kinase of interest in the generation of this phosphoproteome.…”