A high-resolution HPLC-QqTOF platform using parallel reaction monitoring for in-depth lipid discovery and rapid profiling

Abstract

Here, we developed a robust lipidomics workflow merging both targeted and untargeted approaches on a single liquid chromatography coupled to quadrupole-time of flight (LC-QqTOF) mass spectrometry platform with parallel reaction monitoring (PRM). PRM assays integrate both untargeted profiling from MS1 scans and targeted profiling obtained from MS/MS data. This workflow enabled the discovery of more than 2300 unidentified features and identification of more than 600 lipid species from 23 lipid classes at the level of fatty acid/long chain base/sterol composition in a barley root extracts. We detected the presence of 142 glycosyl inositol phosphorylceramides (GIPC) with HN(Ac)-HA as the core structure of the polar head, 12 cardiolipins and 17 glucuronosyl diacylglycerols (GlcADG) which have been rarely reported previously for cereal crops. Using a scheduled algorithm with up to 100 precursors multiplexed per duty cycle, the PRM assay was able to achieve a rapid profiling of 291 species based on MS/MS data by a single injection. We used this novel approach to demonstrate the applicability and efficiency of the workflow to study salt stress induced changes in the barley root lipidome. Results show that 221 targeted lipids and 888 unknown features were found to have changed significantly in response to salt stress. This combined targeted and untargeted single workflow approach provides novel applications of lipidomics addressing biological questions.

Insights Into Oxidized Lipid Modification in Barley Roots as an Adaptation Mechanism to Salinity Stress

“…In recent years, lipidomics has emerged as an effective tool to understand the diversity of membrane lipid compositions and unravel the roles of lipids in plant adaptation and tolerance to abiotic stresses (Welti et al, 2007;Natera et al, 2016;Tenenboim et al, 2016). Lipids and their related metabolites can now be readily detected from cultured cells or tissue extracts, identified and quantified on a large scale with high sensitivity using the latest techniques in lipidomics (Horn and Chapman, 2012;Köfeler et al, 2012;Haslam and Feussner, 2017;Yu et al, 2018). The detected changes in individual lipid species in stress-treated varieties can indicate the functions of lipids and related proteins or genes affecting lipid metabolism or lipid signaling (Welti et al, 2007).…”

“…The detected changes in individual lipid species in stress-treated varieties can indicate the functions of lipids and related proteins or genes affecting lipid metabolism or lipid signaling (Welti et al, 2007). Previous studies on lipid composition alterations in response to various plant abiotic stresses, including drought, wounding, freezing, salinity, and nutrient deficiency, have uncovered diverse roles of membrane lipids and associated genes (Horn and Chapman, 2012;Vu et al, 2014;Tarazona et al, 2015;Natera et al, 2016;Sarabia et al, 2018;Tawaraya et al, 2018;Yu et al, 2018).…”

Comparative spatial lipidomics analysis reveals cellular lipid remodelling in different developmental zones of barley roots in response to salinity

“…The solvent was collected in a new 2.0‐ml tube and dried in a vacuum evaporator. The pellet was resuspended in 200 μL of methanol:butanol (1:1, v/v) containing 20 mM of ammonium acetate of which 10 μL were subjected to LC‐MS/MS analysis (Yu et al, ). As many lipid classes can differently ionize in either electrospray ionization+ or electrospray ionization− using LC‐MS, we analysed lipid extracts obtained from dissected root sections consecutively in both positive and negative ionization modes.…”

“…The LC‐MS/MS data were processed using MultiQuant™ 3.0.2 Software (SCIEX; Framingham, MA, USA), identified using an in‐house‐generated lipid database for barley (Yu et al, ). The data were normalized to the sample fresh weight.…”

“…Although many studies exist on the effects of salinity on crop growth, few have investigated root metabolic profiles (Annunziata et al, 2017;Cao, Lutz, Hill, Callahan, & Roessner, 2016;Gavaghan et al, 2011;Geilfus et al, 2015;Guo et al, 2017;Jiao et al, 2018;Richter, Erban, Kopka, & Zörb, 2015), and an even smaller number have conducted lipidomics analyses of salt-stressed root tissues (Natera, Hill, Rupasinghe, & Roessner, 2016;Yu et al, 2018). Further, most of these comparative studies are based on bulked tissue extractions that often mask tissue-specific metabolic differences.…”