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

Hilaire Yam Fung Cheung,Mailin Manke,Robert Ahrends,Philipp Westhoff,Meinrad Gawaz,Steve P Watson,Oliver Borst,Cristina Coman,Albert Sickmann,Johan W M Heemskerk

doi:10.1021/acs.jproteome.1c00017

Abstract

Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography–mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides.

Highlights

Phosphoinositides are one of the highly diverse glycerophospholipid subcategories
We present here an improved quantitative ion chromatography (IC)-methods using tandem mass spectrometry (MS/MS) workflow for phosphoinositides analysis, which includes the addition of standards, a modified extraction and deacylation procedure, and an optimized IC method, resulting in a potential, −150 V; entrance potential, −10 V; and exit potential, −10 V
For scheduled selected reaction monitoring (SRM), Q1 and Q3 were set to unit resolution

Summary

■ INTRODUCTION

Phosphoinositides are one of the highly diverse glycerophospholipid subcategories. Reversible phosphorylation of the myoinositol headgroup of phosphatidylinositol gives rise to seven distinct phosphoinositides positional isomers in biological systems, namely, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns[3,4]P2, PtdIns[3,5]P2, PtdIns[4,5]P2, and PtdIns[3,4,5]P3. After the addition of 242 μL of CHCl3, 484 μL of MeOH, 23.6 μL of 1 M HCl, 170 μL water, and internal standard (100 pmol of PtdIns[4,5]P2-FP) to the cell pellets containing 1 × 108 platelets, the mixture was allowed to stand at room temperature for 5 min with occasional vortexing. 725 μL of CHCl3 and 170 μL of 2 M HCl were added to induce phase separation and the samples were centrifuged at 1500g for 5 min at room temperature (Eppendorf, Hamburg, Germany). This created a two-phase system with an upper aqueous layer and a protein interface.

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES