Targeted Phosphoinositides Analysis Using High-Performance Ion Chromatography-Coupled Selected Reaction Monitoring Mass Spectrometry

Cheung, Hilaire Yam Fung; Coman, Cristina; Westhoff, Philipp; Manke, Mailin Christin; Sickmann, Albert; Gawaz, Meinrad; Watson, Steve P.; Ahrends, Robert

doi:10.1021/acs.jproteome.1c00017

Cited by 11 publications

(7 citation statements)

References 45 publications

(93 reference statements)

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“…In addition, antibodies should recognize small differences of distinct PI species. Other methods have been developed and used to detect PIs in resting and activated platelets, such as mass spectrometry [31,32]. They accurately quantitatively measure PIs; however, they lack information on the spatial distribution of PIs as well as recognition of potential diverse cellular populations having different PI levels.…”

Section: Discussionmentioning

confidence: 99%

“…They accurately quantitatively measure PIs; however, they lack information on the spatial distribution of PIs as well as recognition of potential diverse cellular populations having different PI levels. In resting PLTs, only three PIs were detected by ion chromatography coupled to tandem mass spectrometry (IC-MS/MS), PI4P, PI(4,5)P 2 (both present at high levels) and PI(3,4)P 2 [32]. Collagen-related peptide (CRP) activation of PLTs caused the increase of all three PIs with the highest change in PI(3,4)P 2 ; however, the kinases or phosphatases responsible for these changes were not studied.…”

Section: Discussionmentioning

confidence: 99%

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Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Bura

Begonja

2021

Life

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Phosphoinositides (PIs) are phosphorylated membrane lipids that have a plethora of roles in the cell, including vesicle trafficking, signaling, and actin reorganization. The most abundant PIs in the cell are phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-4-monophosphate (PI4P). The localization and roles of both PI(4,5)P2 and PI4P are well established, is the broadly accepted methodological approach for their immunocytochemical visualization in different cell compartments in several cell lines. However, not much is known about these PIs in platelets (PLTs), the smallest blood cells that detect vessel wall injury, activate, and stop the bleeding. Therefore, we sought to investigate the localization of PI(4,5)P2 and PI4P in resting and activated PLTs by antibody staining. Here, we show that the intracellular pools of PI(4,5)P2 and PI4P can be detected by the established staining protocol, and these pools can be modulated by inhibitors of OCRL phosphatase and PI4KIIIα kinase. However, although resting PLTs readily stain for the plasma membrane (PM) pools of PI(4,5)P2 and PI4P, just a few activated cells were stained with the established protocol. We show that optimized protocol allows for the visualization of PI(4,5)P2 and PI4P at PM in activated PLTs, which could also be modulated by OCRL and PI4KIIIα inhibitors. We conclude that PI(4,5)P2 and PI4P are more sensitive to lipid extraction by permeabilizing agents in activated than in resting human PLTs, which suggests their different roles during PLT activation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Bura

Begonja

2021

Life

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“…Because of the low abundance of phosphoinositides, this approach requires prior metabolic labelling of cells with radioactive 32 P. More recently, sensitive label-free mass spectrometry-based approaches have come into focus for measuring phosphatidylinositol 3,4,5-trisphosphate 214 and phosphatidylinositol 3,4-bisphosphate (ref. 215 ), and variations on these methods for the global label-free analysis of phosphoinositides have since been reported 216 , 217 .…”

Section: Introductionmentioning

confidence: 99%

Phosphoinositides as membrane organizers

Posor

Jang

Haucke

2022

Nat Rev Mol Cell Biol

188

128

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Phosphoinositides are signalling lipids derived from phosphatidylinositol, a ubiquitous phospholipid in the cytoplasmic leaflet of eukaryotic membranes. Initially discovered for their roles in cell signalling, phosphoinositides are now widely recognized as key integrators of membrane dynamics that broadly impact on all aspects of cell physiology and on disease. The past decade has witnessed a vast expansion of our knowledge of phosphoinositide biology. On the endocytic and exocytic routes, phosphoinositides direct the inward and outward flow of membrane as vesicular traffic is coupled to the conversion of phosphoinositides. Moreover, recent findings on the roles of phosphoinositides in autophagy and the endolysosomal system challenge our view of lysosome biology. The non-vesicular exchange of lipids, ions and metabolites at membrane contact sites in between organelles has also been found to depend on phosphoinositides. Here we review our current understanding of how phosphoinositides shape and direct membrane dynamics to impact on cell physiology, and provide an overview of emerging concepts in phosphoinositide regulation.

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“…Activation of both, GPCR and GPVI signaling pathways, result in a phospholipase C (PLC)-dependent hydrolysis of phosphoinositides (PI) [ 20 ] which is followed by the generation of inositol-1,4,5-triphosphate (IP 3 ) and 1,2-diacyl-glycerol (DAG) [ 7 ]. While GPCRs utilize the PLC-ß isoform, the GPVI-dependent signaling pathway acts via the PLC-γ isoform.…”

Section: Platelet Physiology and Mechanisms/pathways Of Ca ...mentioning

confidence: 99%

CRACking the Molecular Regulatory Mechanism of SOCE during Platelet Activation in Thrombo-Occlusive Diseases

Münzer

2022

Cells

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Thrombo-occlusive diseases such as myocardial infarction, ischemic stroke and deep vein thrombosis with subsequent pulmonary embolism still represent a major health burden worldwide. Besides the cells of the vasculature or other hematopoietic cells, platelets are primarily responsible for the development and progression of an occluding thrombus. The activation and function of platelets crucially depend on free cytosolic calcium (Ca2+) as second messenger, which modulates platelet secretion, aggregation and thrombus formation. Ca2+ is elevated upon platelet activation by release of Ca2+ from intracellular stores thus triggering of the subsequent store-operated Ca2+ entry (SOCE), which is facilitated by Ca2+ release-activated channels (CRACs). In general, CRACs are assembled by the pore-forming unit Orai in the plasma membrane and the Ca2+-sensing stromal interaction molecule (STIM) in the endoplasmic reticulum after the depletion of internal Ca2+ stores. In the last few years, there is a growing body of the literature demonstrating the importance of STIM and Orai-mediated mechanism in thrombo-occlusive disorders. Thus, this review provides an overview of the recent understanding of STIM and Orai signaling in platelet function and its implication in the development and progression of ischemic thrombo-occlusive disorders. Moreover, potential pharmacological implications of STIM and Orai signaling in platelets are anticipated and discussed in the end.

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Targeted Phosphoinositides Analysis Using High-Performance Ion Chromatography-Coupled Selected Reaction Monitoring Mass Spectrometry

Cited by 11 publications

References 45 publications

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Imaging of Intracellular and Plasma Membrane Pools of PI(4,5)P2 and PI4P in Human Platelets

Phosphoinositides as membrane organizers

CRACking the Molecular Regulatory Mechanism of SOCE during Platelet Activation in Thrombo-Occlusive Diseases

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