A Targeted, Bioinert LC–MS/MS Method for Sensitive, Comprehensive Analysis of Signaling Lipids

Signaling lipids are key players in cellular processes. Despite their importance, no method currently allows their comprehensive monitoring in one analytical run. Challenges include a wide dynamic range, isomeric and isobaric species, and unwanted interaction along the separation path. Herein, we present a sensitive and robust targeted liquid chromatography-mass spectrometry (LC–MS/MS) approach to overcome these challenges, covering a broad panel of 17 different signaling lipid classes. It involves a simple one-phase sample extraction and lipid analysis using bioinert reversed-phase liquid chromatography coupled to targeted mass spectrometry. The workflow shows excellent sensitivity and repeatability in different biological matrices, enabling the sensitive and robust monitoring of 388 lipids in a single run of only 20 min. To benchmark our workflow, we characterized the human plasma signaling lipidome, quantifying 307 endogenous molecular lipid species. Furthermore, we investigated the signaling lipidome during platelet activation, identifying numerous regulations along important lipid signaling pathways. This highlights the potential of the presented method to investigate signaling lipids in complex biological systems, enabling unprecedentedly comprehensive analysis and direct insight into signaling pathways.


Platelets
Mice were anesthetized and blood was drawn from the retrobulbar plexus into citrate anticoagulated tubes.Platelet-rich plasma (PRP) was obtained by centrifuging at 264 g for 5 min followed by 52 g for 6 min.Afterwards, PRP was centrifuged at 640 g for 5 min to pellet the platelets.After a washing step, the obtained platelet pellet was resuspended in modified Tyrode-HEPES buffer, pH 7.4 (137 mM NaCl, 12 mM NaHCO3, 2 mM KCl, 5.5 mM glucose, 0.3 mM NaH2PO4, 5 mM HEPES, 1 mM CaCl2).The cell count was determined using a Sysmex automatic hematology analyzer (Norderstedt, Germany), with one sample equaling 10 8 platelets.The freshly isolated platelets were then either left in a resting state or stimulated with 5 µg/mL CRP and 1 U/mL thrombin for 5 min.The pellet and releasate were separated by centrifuging for 5 min at 640 g and RT and subsequently separately shock frozen in liquid nitrogen and stored at -80 °C until further use.All animal experiments were performed according to the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by local authorities (Regierungspräsidium Tübingen) following the ARRIVE guidelines.

Plasma
Blood was drawn into 3.8% sodium citrate tubes and centrifuged at 1,000 g and 4 °C for 10 min.The

BuEt two-phase extraction
5 µL IS mix and 350 µL 200 mM citric acid + 100 mM K2HPO4 (pH 4.5) were added to 100 µL thawed plasma. 1 mL BuOH/EtOAc 1/1 (v/v) was added and the sample was thoroughly mixed using a vortex mixer and subsequently ultrasonicated on ice for 10 s.After vigorous mixing for another 2 min, the sample was centrifuged at 4 °C and 21,000 g for 10 min.900 µL of the upper organic phase were collected and the lower phase was re-extracted using 400 µL water-saturated BuOH and 400 µL EtOAc.
The combined organic phases were dried under a nitrogen stream.

BuMe one-phase extraction
5 µL IS mix and 1 mL BuOH/MeOH 3/1 (v/v) were added to 100 µL thawed plasma.The sample was thoroughly mixed using a vortex mixer and subsequently ultrasonicated on ice for 10 s.After mixing for another 10 s, the sample was incubated at 4 °C and 950 rpm for 30 min.The protein fraction was precipitated by centrifuging at 4 °C and 21,000 g for 15 min.The organic phase was recovered and dried under a nitrogen stream.

MMC one-phase extraction
5 µL IS mix and 1 mL MeOH/MTBE/CHCl3 1.33/1/1 (v/v/v) were added to 100 µL thawed plasma.The sample was thoroughly mixed using a vortex mixer and subsequently ultrasonicated on ice for 10 s.
After mixing for another 10 s, the sample was incubated at 4 °C and 950 rpm for 30 min.The protein fraction was precipitated by centrifuging at 4 °C and 21,000 g for 15 min.The organic phase was recovered and dried under a nitrogen stream.

Figure S1
Figure S1 Influence of different column coatings on the separation of the critical pair PGE2/PGD2.

Figure S2
Figure S2Chromatographic separation of (A) lysoglycerophospholipids and (B) the critical oxylipin pair PGE2/PGD2 in plasma.Separation of CPA and LPS from LPA is crucial due to possible in-source fragmentation.Furthermore, PGE2/PGD2 also need to be chromatographically separated, as they do not possess unique MS/MS fragments.

Figure
Figure S3Equivalent carbon number (ECN) model plots.

Figure S8
Figure S8Retention time stability of the LC-MS/MS method over multiple weeks and different matrices for selected analytes (n=188).

Figure S10
Figure S10 Analysis of the signaling lipidome in platelets during platelet activation (n=5).Platelets were stimulated using 1 U/mL thrombin and 5 μg/mL CRP for 5 min.(A) Lipid spaces illustrating the regulation of the signaling lipidome in platelet and their releasates during platelet activation.(B) Bar graph depicting regulated and non-regulated lipids in the platelet releasate upon stimulation.(B) Quantitative lipid changes of different lipid categories in the platelet releasate during platelet activation