A simple and universal method for the separation and identification of phospholipid molecular species

Abstract

One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a simple yet universal high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method to separate molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid profiling using full scan mass spectrometry, as well as lipid class specific scanning in positive and negative ionisation mode. This allows high-throughput processing of samples for lipidomics, even if different types of MS analysis are required. Excellent separation of isobaric and even isomeric species is achieved, and original levels of lyso-lipids can be determined without interference from lyso-lipids formed from diacyl species by source fragmentation. As examples of application of this method, more than 400 phospholipid species were identified and quantified in crude phospholipid extracts from rat liver and the parasitic helminth Schistosoma mansoni.

SCAP is required for timely and proper myelin membrane synthesis

“…Before infusion, neutral lipids were separated on a Lichrospher RP18e column by using a gradient from acetonitrile to acetone. Phospholipids, glycosphingolipids, and free fatty acids obtained by hydrolysis of phospholipids were analyzed as described (30,31) by using defined molecular species and authentic free fatty acid standards as standards for quantification purposes.…”

Lysophospholipids secreted by splenic macrophages induce chemotherapy resistance via interference with the DNA damage response

“…Protein content was measured using a spectophotometer and control western blots were performed. DNA, nuclear and cytoplasmic fractions were hydrolyzed and analyzed by inductively coupled plasma mass spectrometry [22][23][24] .…”

“…Total lipids from culture media of sCM were extracted using a modified Bligh & Dyer extraction according to the study by Retra et al 24 Total lipid fraction was separated into four fractions (neutral lipids), free fatty acids, two phospholipid fractions (PC/PE/SM and PI/PS/SM) by solid phase extraction according to the study by Kim et al 22 The PC/ PE/SM fraction was further fractionated into separate lipid classes by hydrophilic interaction liquid chromatography as described by the study by Brouwers et al 23 The HPLC output was split into two flows (1:10 ratio), of which one was lead to a mass spectrometer to monitor the separation process, the other major flow was used to collect lipid classes. LPC analysis was performed using a modified method by Retra et al 24 Here the HPLC column was replaced by a fused core HALO C18 column (Biotech, Onsala, Sweden). To increase sensitivity, LPCs were detected in multiple reaction monitoring mode, in which a collection of 36 different LPC masses (14:0 to 26:6 LPC) were monitored and confirmed by the formation of a phosphocholine fragment of 184 m/z (collision energy was 55 V).…”

Plackett-Burman Design and Fragmentation Studies to Assist the Comparison of Techniques used to Extract Phospholipids Prior to Regiospecific Characterization by Liquid Chromatography Mass Spectrometry

“…The gradient program was as follows: the initial condition was set at 40% B for 9 min, ramped in 15 min to 100% B, where it was kept for 135 min, and returned to 40% B in 1 min. The ESI and MS conditions were set according to It must be mentioned that the mobile phase composition was adapted from an article published elsewhere (Retra, et al, 2008), where ammonium acetate and L-serine were used as additives. Ammonium acetate promotes the formation of protonated phospholipid molecular species in the ESI-MS (Zeng et al, 2013).…”