Development and Application of Multidimensional Lipid Libraries to Investigate Lipidomic Dysregulation Related to Smoke Inhalation Injury Severity

Abstract

The implication of lipid dysregulation in diseases, toxic exposure outcomes, and inflammation has brought great interest to lipidomic studies. However, lipids have proven to be analytically challenging due to their highly isomeric nature and vast concentration ranges in biological matrices. Therefore, multidimensional techniques such as those integrating liquid chromatography, ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (LC-IMS-CID-MS) have been implemented to separate lipid isomers as well as provide structural information and increased identification confidence. These data sets are however extremely large and complex, resulting in challenges for data processing and annotation. Here, we have overcome these challenges by developing sample-specific multidimensional lipid libraries using the freely available software Skyline. Specifically, the human plasma library developed for this work contains over 500 unique lipids and is combined with adapted Skyline functions such as indexed retention time (iRT) for retention time prediction and IMS drift time filtering for enhanced selectivity. For comparison with other studies, this database was used to annotate LC-IMS-CID-MS data from a NIST SRM 1950 extract. The same workflow was then utilized to assess plasma and bronchoalveolar lavage fluid (BALF) samples from patients with varying degrees of smoke inhalation injury to identify lipid-based patient prognostic and diagnostic markers.

Keywords: data annotation; ion mobility spectrometry; lipidomics; smoke inhalation; spectral libraries.

Figure 1.

Overview of select toxic components of smoke, inhalation injury outcomes, and the sample types studied in this work.

Figure 2.

Conformational space of plasma lipids included in (A) positive and (B) negative ionization mode libraries. All 854 lipid features, corresponding to 516 unique lipids when accounting for multiple ion types, are plotted by CCS versus m/z value. These lipids span 18 classes from five lipid categories. Features are colored by class to emphasize class-specific trendlines.

Figure 3.

Predictive capabilities of the plasma iRT calculator. Accuracy of iRT retention time predictions plotted as predicted versus observed retention times for triplicate BTLE data collected on a second column and instrument with the same mobile phase composition and LC gradient method at (A) the original run time of 38 min and (B) a shortened run time of 14 min. iRT prediction utility in assigning LC-separated isomers such as (C) LPC(0:0/18:1) and (D) LPC(18:1/0:0), as shown by the predicted gray bar.

Figure 4.

Use of sodiated phosphatidylcholines for fatty acyl assignment of PC(18:1_20:4). This example illustrates raw peak areas for (A) protonated and (B) sodiated PC(18:0_20:4) precursor and fragment signals in positive mode as bar charts and peak area percentages as pie charts.

Figure 5.

Drift time filtering for target lipid features in Skyline. Extracted ion chromatogram for the PE(18:0_20:4) precursor, M + 1 and M + 2 isotopes, and three characteristic fragments illustrate how ( A) before drift time filtering there are numerous and (B) following drift time filtering a majority of the peaks are removed since they are not affiliated with the target ion. (C) Fragment DIA drift spectra for FA 18:0 [M − H]⁻, where the signal outside of the purple drift time window belongs to another precursor ion and is filtered out, thereby decreasing interference and improving quantitative capabilities.

Figure 6.

Analysis of lipid significance and associated trends. (A) Volcano plots of the identified lipids where red and blue outlines indicate up- and downregulated lipids, respectively, in subjects whose smoke inhalation injuries led to mortality. (B) Association of subject mortality and the fatty acyl composition of statistically significant TGs. When the number of double bonds is plotted against the sum of fatty acyl carbons, a trend emerges in plasma where TGs with fewer carbons and double bonds are downregulated, and those with longer, more unsaturated fatty acyls are upregulated in subjects whose injuries led to mortality.