Expanding Lipidome Coverage Using LC-MS/MS Data-Dependent Acquisition with Automated Exclusion List Generation

Abstract

Untargeted omics analyses aim to comprehensively characterize biomolecules within a biological system. Changes in the presence or quantity of these biomolecules can indicate important biological perturbations, such as those caused by disease. With current technological advancements, the entire genome can now be sequenced; however, in the burgeoning fields of lipidomics, only a subset of lipids can be identified. The recent emergence of high resolution tandem mass spectrometry (HR-MS/MS), in combination with ultra-high performance liquid chromatography, has resulted in an increased coverage of the lipidome. Nevertheless, identifications from MS/MS are generally limited by the number of precursors which can be selected for fragmentation during chromatographic elution. Therefore, we developed the software IE-Omics to automate iterative exclusion (IE), where selected precursors using data-dependent topN analyses are excluded in sequential injections. In each sequential injection, unique precursors are fragmented until HR-MS/MS spectra of all ions above a user-defined intensity threshold are acquired. IE-Omics was applied to lipidomic analyses in Red Cross plasma and substantia nigra tissue. Coverage of the lipidome was drastically improved using IE. When applying IE-Omics to Red Cross plasma and substantia nigra lipid extracts in positive ion mode, 69 % and 40 % more molecular identifications were obtained, respectively. In addition, applying IE-Omics to a lipidomics workflow increased the coverage of trace species, including odd-chained and short-chained diacylglycerides and oxidized lipid species. By increasing the coverage of the lipidome, applying IE to a lipidomics workflow increases the probability of finding biomarkers and provides additional information for determining etiology of disease.

Structure Annotation and Quantification of Wheat Seed Oxidized Lipids by High-Resolution LC-MS/MS

“…Furthermore, the spectra reveal structural information on the molecular composition of the lipids and positional information of the oxidation. We followed a procedure similar to that described by Koelmel et al (2017), but instead of using exclusion lists, we constructed a scheduled precursor list (SPL) from all potentially monoisotopic peaks from the profiling experiment. The benefit is that MS/MS spectra are collected more effectively, because no MS/MS spectra other than relevant are produced (i.e.…”

Common cases of improper lipid annotation using high-resolution tandem mass spectrometry data and corresponding limitations in biological interpretation

“…It is important to note that most software only include one lipid identification for a given feature in the final report, which is based on the false assumption that there are few instances of co-eluting lipids. Examples of annotated lipids using pipes can be found in Supplementary Table S-4 of Koelmel et al [42], for example for m/z 766.5391 at retention time 7.06, the feature was annotated as PE(18:0_20:5)+H | PE(18:1_20:4)+H | PE(16:0_22:5)+H, with annotations ranked by a score based on the MS/MS spectra.…”

“…In software that implements rule-based identification such as LipidSearch (Thermo Scientific), annotation is based on fragments observed, and lipids will be reported either by lipid class (class, total carbons, and total double bonds), or by fatty acyl constituents. For MS/MS based identification, LipidMatch [42] is the only lipid identification software to date which employs all the annotation guidelines presented here, including using pipes “|” for multiple identifications “_” when fatty acyl position on the glycerol backbone is unknown, and annotates lipids by total carbons and degrees of unsaturation when only class specific fragments are observed. For exact mass searching, LipidPioneer [51], designed as a Microsoft Excel workbook, is the only template where users can generated exact mass libraries that provide exact masses and adducts for lipid species annotated with the slash, “/”, underscore, “_”, or only by class and total carbons and degrees of unsaturation depending on the users end use.…”

A Robust Lipidomics Workflow for Mammalian Cells, Plasma, and Tissue Using Liquid-Chromatography High-Resolution Tandem Mass Spectrometry

“…Software for annotating lipid structure based on frag-mentation for LC-MS/MS data ( see Note 22 ) includes LipidSearch [31, 32], MS-DIAL [22], and LipidMatch [33] or an in-house fragmentation library/software. The lipid identification software, LipidMatch [33], provides additional unique features in comparison to MS-Dial, including the ability to incorporate MZmine and XCMS outputs, to obtain structurally defined lipid assignments, and to mine simulated MS/MS libraries for oxidized lipids. Table 2 provides a list of 31 of the 56 lipid types with in silico fragmentation libraries in LipidMatch for identification of various lipid species.…”