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Sub-5-min RP-UHPLC-TIMS for high-throughput untargeted lipidomics and its application to multiple matrices

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Abstract

Untargeted lipidomics, with its ability to take a snapshot of the lipidome landscape, is an important tool to highlight lipid changes in pathology or drug treatment models. One of the shortcomings of most untargeted lipidomics based on UHPLC-HRMS is the low throughput, which is not compatible with large-scale screening. In this contribution, we evaluate the application of a sub-5-min high-throughput four-dimensional trapped ion mobility mass spectrometry (HT-4D-TIMS) platform for the fast profiling of multiple complex biological matrices. Human AC-16 cells and mouse brain, liver, sclera, and feces were used as samples. By using a fast 4-min RP gradient, the implementation of TIMS allows us to differentiate coeluting isomeric and isobaric lipids, with correct precursor ion isolation, avoiding co-fragmentation and chimeric MS/MS spectra. Globally, the HT-4D-TIMS allowed us to annotate 1910 different lipid species, 1308 at the molecular level and 602 at the sum composition level, covering 58 lipid subclasses, together with quantitation capability covering more than three orders of magnitude. Notably, TIMS values were highly comparable with respect to longer LC gradients (CV% = 0.39%). These results highlight how HT-4D-TIMS-based untargeted lipidomics possess high coverage and accuracy, halving the analysis time with respect to conventional UHPLC methods, and can be used for fast and accurate untargeted analysis of complex matrices to rapidly evaluate changes of lipid metabolism in disease models or drug discovery campaigns.

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Abbreviations

AHexCer:

Acylhexosylceramide

ASM:

Acylsphingomyelin

BMP:

Bismonoacylglycerophosphate

CL:

Cardiolipin

CAR:

Carnitine

Cer:

Ceramide

CE:

Cholesteryl ester

CoQ:

Coenzyme Q

DG:

Diacylglycerol

DGGA:

Diacylglyceryl glucuronide

DGDG:

Digalactosyldiacylglycerol

Hex2Cer:

Dihexosylceramide

DCAE:

Esterified deoxycholic acid

DG O-:

Ether-linked diacylglycerol

LPC O-:

Ether-linked lysophosphatidylcholine

LPE O-:

Ether-linked lysophosphatidylethanolamine

MGDG O-:

Ether-linked monogalactosyldiacylglycerol

Ox-PE-O-:

Ether-linked oxidized phosphatidylethanolamine

PC O-:

Ether-linked phosphatidylcholine

PE O-:

Ether-linked phosphatidylethanolamine

PG O-:

Ether-linked phosphatidylglycerol

PI O-:

Ether-linked phosphatidylinositol

PS O-:

Ether-linked phosphatidylserine

TG O-:

Ether-linked triacylglycerol

FA:

Fatty acid

FAHFA:

Fatty acid ester of hydroxyl fatty acid

GM3:

Ganglioside GM3

HBMP:

Hemibismonoacylglycerophosphate

HexCer:

Hexosylceramide

LPC:

Lysophophatidylcholine

LPE:

Lysophosphatidylethanolamine

LPG:

Lysophosphatidylglycerol

LPI:

Lysophosphatidylinositol

LPS:

Lysophosphatidylserine

MG:

Monoacylglycerol

MGDG:

Monogalactosyldiacylglycerol

NAGly:

N-Acyl glycine

NAGlySer:

N-Acyl glycyl serine

Ox-PI-Cer:

Oxidized ceramide phosphoinositol

Ox-PE:

Oxidized phosphatidylethanolamine

Ox-PI:

Oxidized phosphatidylinositol

Ox-SM:

Oxidized sphingomyelin

Ox-SHexCer:

Oxidized sulfatide

PC:

Phosphatidylcholine

PEtOH:

Phosphatidylethanol

PE:

Phosphatidylethanolamine

PG:

Phosphatidylglycerol

PI:

Phosphatidylinositol

PMeOH:

Phosphatidylmethanol

PS:

Phosphatidylserine

SPB:

Sphingoid bases

SM:

Sphingomyelin

ST:

Sterol

SE:

Sterol ester

SHexCer:

Sulfatide

TG:

Triacylglycerol

Hex3Cer:

Trihexosylceramide

Vit. D:

Vitamin D

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Acknowledgements

Cells and tissues were kindly donated as part of secondary experiments from Prof. Carrizzo the Department of Medicine (DIPMED, University of Salerno, Italy) and from Prof. Paolisso of the Department of Advanced Medical and Surgical Sciences, University of Campania “Luigi Vanvitelli”, Naples, Italy.

Funding

This work was supported by Ministero dell’Università e della Ricerca (MIUR) project PIR01_00032 BIO OPEN LAB BOL “CUP” J37E19000050007, project CIR01_00032 – BOL “BIO Open Lab—Rafforzamento del capitale umano” and project PNC0000001 D34 Health—Digital Driven Diagnostics, prognostics and therapeutics for sustainable Health care “CUP” B53C22006090001 and Regione Campania (Italy) grant “Combattere la resistenza tumorale: piattaforma integrata multi-disciplinare per un approccio tecnologico innovative alle oncoterapie—CAMPANIA ONCO-TERAPIE” project number B61G18000470007. to P. Campiglia.

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Author notes

  1. Fabrizio Merciai and Manuela Giovanna Basilicata equally contributed as first author.

Authors and Affiliations

  1. Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 13284084, Fisciano, SA, Italy

    Fabrizio Merciai, Manuela Giovanna Basilicata, Danila La Gioia, Emanuela Salviati, Vicky Caponigro, Tania Ciaglia, Simona Musella, Carlo Crescenzi, Eduardo Sommella & Pietro Campiglia

  2. PhD Program in Drug Discovery and Development, University of Salerno, Fisciano, SA, Italy

    Danila La Gioia

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  1. Fabrizio Merciai
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Contributions

Conceptualization: FM. Methodology: VC. Formal analysis and investigation: DLG, ES. Data curation: TC. Writing—original draft preparation: MGB. Writing—review and editing: ES, SM. Funding acquisition: PC. Supervision: ES, CC.

Corresponding author

Correspondence to Eduardo Sommella.

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Ethics approval

The study was conducted in conformity with Italian (D.L. 26/2014) and European (directive 2010/63/EU) regulations on the protection of animals used for scientific purposes and approved by the Italian Ministry.

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Merciai, F., Basilicata, M.G., La Gioia, D. et al. Sub-5-min RP-UHPLC-TIMS for high-throughput untargeted lipidomics and its application to multiple matrices. Anal Bioanal Chem 416, 959–970 (2024). https://doi.org/10.1007/s00216-023-05084-w

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