Phosphoproteomics in the Age of Rapid and Deep Proteome Profiling

Riley, Nicholas M.; Coon, Joshua J.

doi:10.1021/acs.analchem.5b04123

Cited by 238 publications

(274 citation statements)

References 286 publications

(470 reference statements)

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“…The field of phosphoproteomics has long been concerned with deep coverage of protein phosphorylation, 69, 216, 217 but while these broad surveys are incredibly important, they are still essentially snapshots taken at different points during the cellular cycle. It is possible to identify tens of thousands of phosphorylation sites, especially with extensive pre-fractionation, but issues with stochastic sampling remain in shotgun experiments, and the choice of protease, enrichment strategy, quantitation approach, and sample storage method can still introduce slight bias.…”

Section: Future Perspectivesmentioning

confidence: 99%

Recent advances in phosphoproteomics and application to neurological diseases

Arrington

Hsu

Elder

et al. 2017

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Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

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Section: Future Perspectivesmentioning

confidence: 99%

Recent advances in phosphoproteomics and application to neurological diseases

Arrington

Hsu

Elder

et al. 2017

Analyst

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“…The breakthrough in the technical development, which enabled the use of HPLC-MS/MS as a comprehensive revelation engine for proteins and peptides, was the invention of soft ionization techniques such as ESI (electrospray ionization) [50], which enable the direct mass spectrometric analysis of biological samples from liquid, often aqueous solutions. Nowadays, mass spectrometry is the primary identification and quantification tool for comprehensive phosphoproteomics [51, 52]. Moreover, identification technologies based upon the gas-phase fragmentation of peptide ions [53] and the matching of the resulting set of fragment ions with protein sequence databases [54–56] have laid the ground for the high-throughput identification and quantification of proteins in proteomic samples, enabling the analysis of more than 10,000 proteins in a single 12-day experiment [57].…”

Section: Phosphoproteome Analysis Of Cancer and Cancer Stem Cellsmentioning

confidence: 99%

“…Since the complexity of the cellular proteome hinders the direct analysis of phosphopeptides that are usually present in concentrations much lower than their non-phosphorylated analogues, further fractionation and phosphopeptide enrichment is needed to investigate the phosphoproteome. Different enrichment and fractionation methods have been applied, which were recently reviewed [51]. Typically, enrichment strategies rely on affinity chromatography taking advantage of the phosphate-specific binding abilities of certain metal oxides [60] (titanium dioxide, tin oxide [61]) or of immobilized metal ions such as Fe 3+ [62] or Ga 3+ [63].…”

Section: Hplc-ms/ms Workflow For Phosphoproteomicsmentioning

confidence: 99%

Understanding cell signaling in cancer stem cells for targeted therapy – can phosphoproteomics help to reveal the secrets?

et al. 2017

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BackgroundCancer represents heterogeneous and aberrantly proliferative manifestations composed of (epi)genetically and phenotypically distinct cells with a common clonal origin. Cancer stem cells (CSC) make up a rare subpopulation with the remarkable capacity to initiate, propagate and spread a malignant disease. Furthermore, CSC show increased therapy resistance, thereby contributing to disease relapse. Elimination of CSC, therefore, is a crucial aim to design efficacious treatments for long-term survival of cancer patients. In this article, we highlight the nature of CSC and propose that phosphoproteomics based on unbiased high-performance liquid chromatography-mass spectrometry provides a powerful tool to decipher the molecular CSC programs. Detailed knowledge about the regulation of signaling processes in CSC is a prerequisite for the development of patient-tailored multi-modal treatments including the elimination of rare CSC.Main bodyPhosphorylation is a crucial post-translational modification regulating a plethora of both intra- and intercellular communication processes in normal and malignant cells. Small-molecule targeting of kinases has proven successful in the therapy, but the high rates of relapse and failure to stem malignant spread suggest that these kinase inhibitors largely spare CSC. Studying the kinetics of global phosphorylation patterns in an unbiased manner is, therefore, required to improve strategies and successful treatments within multi-modal therapeutic regimens by targeting the malignant behavior of CSC. The phosphoproteome comprises all phosphoproteins within a cell population that can be analyzed by phosphoproteomics, allowing the investigation of thousands of phosphorylation events. One major aspect is the perception of events underlying the activation and deactivation of kinases and phosphatases in oncogenic signaling pathways. Thus, not only can this tool be harnessed to better understand cellular processes such as those controlling CSC, but also applied to identify novel drug targets for targeted anti-CSC therapy.ConclusionState-of-the-art phosphoproteomics approaches focusing on single cell analysis have the potential to better understand oncogenic signaling in heterogeneous cell populations including rare, yet highly malignant CSC. By eliminating the influence of heterogeneity of populations, single-cell studies will reveal novel insights also into the inter- and intratumoral communication processes controlling malignant CSC and disease progression, laying the basis for improved rational combination treatments.

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“…Although a specific function for WDY in spermatogenesis has yet to be 389 determined, WDY is expressed in a testis-specific manner and under positive selection in the D. 390 melanogaster group [58]. The relative paucity of phosphorylation PTMs may reflect the fact that 391 phosphorylation is one of the more difficult PTMs to identify with certainty via mass spectrometry based 392 proteomics [59]. However, it is also noteworthy that sperm samples in this study were purified from the 393 male seminal vesicle, and thus, before transfer to the female reproductive tract.…”

Section: Post-translational Modification Of Sperm Proteins 373mentioning

confidence: 99%