Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry

Abstract

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.

Low colostrum intake results in potential accumulation of peroxisome lipid substrates in vaginal tissue of 3-week-old gilts

“…Another distinct analysis of phosphatidylcholine and sphingomyelin classes (PCSM) of lipids was also run. The attributions are tentative and based on the Lipid Maps database ( Reis et al, 2023 ). Lipids that distinguished between low and high IM and low and high 24 h weight gain are listed in Table 1 .…”

“…Relative ion intensity was determined by dividing the intensity by the sum of intensities of all lipids within a sample, and within a screening method. Lipid Maps ( ) was used to attribute tentative lipid classes based on functional group and biological information ( Reis et al, 2023 ). Relative intensity ion pairs were uploaded to Metaboanalyst 5.0 and the data was normalized using the auto scaling feature.…”

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes

“…A capillary pump was connected to the autosampler and operated at a flow rate of 10 µL/min and a pressure of 150 bar. The capillary voltage on the instrument was 5kV and the gas flow was 5.1 L/min at 300 • C. The multiple reaction monitoring (MRM) profiling approach was utilized to evaluate the lipid components of the macrophage lysosomes using the recently reported methods [28,29]. The Equisplash Lipidomics Internal Standard, Avanti p/n 330731 (1 ng of each lipid in the mix), was used as a quality control sample to monitor the instrument performance.…”