Phosphoproteome Analysis

Abstract

Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.

Posttranslational Modifications of Rad51 Protein and Its Direct Partners: Role and Effect on Homologous Recombination – Mediated DNA Repair

“…On the contrary, the peptide containing Tyrosine 315 is 28 amino acids long and its pHi is 4,03. Thus, the Y315 peptide is longer and more negatively charged compared to the Y54 peptide which could interfere with its detection by mass spectroscopy (Raggiaschi et al, 2005).…”

Huntington's disease: from experimental results to interaction networks, patho-pathway construction and disease hypothesis

“…Cell extracts were prepared at 0, 12, and 48 hours post-induction to identify proteins involved in pre-apoptotic intracellular events. Protein expression modulation was measured using DIGE technology [2] followed by statistical analysis for spot selection and automated spot picking. The protein content of each picked spot was analyzed by mass spectrometry (MS).…”

Oncoproteomic Approaches in Lung Cancer Research

“…To isolate and identify phosphorylated proteins one must use immunoaffinity or immunoprecipitation with a specific antibody, chromatofocusing, ion exchange chromatography and affinity chromatography, such as immobilized metal ion affinity chromatography (IMAC) (49). Separation methods include electrophoresis, 2D-PAGE or 2D-DIGE coupled with phosphoprotein staining (Pro-Q Diamond phosphoprotein gel stain) or isotope labelling (ICAT, SILAC) (50,51). Analysis and identification methods of phosphoproteins and phosphopeptides are mass spectrometry-based approaches, such MALDI-TOF MS, LC-ESI-MS and MS/MS (52).…”