Recent findings and technological advances in phosphoproteomics for cells and tissues

Abstract

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

An emerging role for the ribosome as a nexus for post-translational modifications

“…Recent technological advancement in phosphoproteomic methods that combine strong cation exchange chromatography with phospho-peptide enrichment methods has increased the dynamic range and sensitivity for the identification of phosphoproteins [16]. Using these techniques, many groups have investigated phosphorylation sites proteome-wide, demonstrating changes during stem cell differentiation, across different mouse tissues, and during difference stages of the mammalian cell cycle [18–20].…”

“…However, PTM assignment based on mass difference, such as acetylation or methylation, is not comprehensive and is prone to misinterpretation as specific masses could result from a combination of multiple PTMs or could be due to artifacts arising from sample preparation methods. Recent advances in PTM enrichment methods from digested peptides, combined with high-resolution quantitative mass spectrometry (MS), permit systems-level PTM mapping for phosphorylation and ubiquitylation, whereby the conjugation sites of the modification can also be identified with higher confidence [16,17]. Interestingly, many of these near-comprehensive static PTM maps or dynamic PTM snapshots in response to different perturbations contain RPs as candidates [18–22,23••].…”

Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

“…Thus, an integrative approach to elucidating the molecular events evoked by DON in IECs is urgently needed. Recently, mass spectrometry (MS)-based phosphoproteomic methods allowing large-scale identification and quantification of phosphoproteins have been employed to uncover specific signaling events in diverse biological processes [17]. The changes in the proteome and phosphoproteome caused by DON have been previously examined in macrophages, and the immunotoxicity of this compound is well documented [18,19].…”

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells

“…Advances in phosphopeptide enrichment methods and MS analysis, together with improved data analysis tools, have made it possible to identify thousands of phosphoproteins from cellular samples (reviewed in [10,11]). Phosphoproteome studies on host responses to viral infection [12][13][14][15][16] have shown that viral infection alters the phosphorylation status of hundreds of proteins involved in pathways critical to the host response to infection.…”