Evaluation of the Performance of Lipidyzer Platform and Its Application

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Evaluation of the Performance of Lipidyzer Platform and its Application in the Lipidomics Analysis in Mouse Heart and Liver Zhijun Cao, Thomas C Schmitt, Vijayalakshmi Varma, Daniel Sloper, Richard D Beger, and Jinchun Sun J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00289 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Journal of Proteome Research

Evaluation of the Performance of Lipidyzer Platform and its Application in the Lipidomics Analysis in Mouse Heart and Liver Zhijun Cao1, Thomas C. Schmitt1, Vijayalakshmi Varma1, Daniel Sloper1, Richard D. Beger1, Jinchun Sun1‫٭‬ 1Division

of System Biology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR, USA

*Corresponding author: Division of Systems Biology, National Center for Toxicological Research, US FDA, 3900 NCTR Road, Jefferson, AR, 72079, USA. E-mail: [email protected], Telephone: +1 870-543-7556, Fax: +1 870-543-7686.

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Abstract Lipids play important roles in cell signaling, energy storage, and as major structural components of cell membranes. To date, little work has been conducted to show the extent of tissue specificity of lipid compositions. Here, the recently acquired Lipidyzer platform was employed in this pilot study: i) to assess the performance of the Lipidyzer platform, ii) to explore lipid profiles in liver and cardiac tissue in mice, iii) to examine sex-specific differences in lipids in the liver tissue, and iv) to evaluate biological variances in lipidomes present in animals. In total, 787 lipid species from 13 lipid classes were measured in the liver and heart. Lipidomics data from the Lipidyzer platform was very reproducible with the coefficient of variations of the quality control (QC) samples ~10%. The total concentration of the cholesterol esters (CE) lipid class, and specifically CE(16:1) and CE(18:1) species showed sex differences in the liver. Cardiac tissue had higher levels of phospholipids containing docosahexaenoic acid, which could be related to heart health status and function. Our results demonstrate the usefulness of the Lipidyzer platform in identifying differences in lipid profile at the tissue level and between male and female mice in specific tissues. Keywords: Lipidyzer, lipidomics, mouse lipid profiling, lipid distribution, tissue-specific lipid profiles


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INTRDUCTION Lipids are the fourth major class of biomolecules, ranking after proteins, carbohydrates and nucleotides. Lipids are known of having widely varied structures. It is estimated that a biological system contains >100,000 different lipid species, which can be categorized into classes based on their structural characteristics; each class contains several lipid species that vary in their fatty acyl chain lengths and the extent of unsaturation in terms of the number and position of double bonds 1, 2. In addition, cellular lipids have wide concentration ranges from attomol to nmol/mg protein and their concentrations are dependent on the biological environment and physiological and health status of the organism or individual. These factors make it challenging to accurately identify lipid biomarkers of disease and toxicity and quantitate lipid species in biological samples. Lipidomics is defined as “the large-scale study of pathways and networks of cellular lipids in biological systems” 1, while the lipidome (a subset of the metabolome) is used to describe the complete lipid profile within a cell, tissue, organism, or a biosystem. In the last decade, the emerging field of lipidomics has been greatly advanced due to rapid developments in liquid chromatography (LC) and mass spectrometry (MS) 3, 4, which makes the detection and quantitation of the lipidome possible with a single platform 5. There are two primary types of lipid analysis, shotgun and LC/MS-based lipidomics. Many reviews of lipidomics have covered the techniques employed as well as its applications in scientific research 6-9. However, none of the reviews cover the newly developed Lipidyzer Platform (SCIEX), which employs direct infusion, differential mobility spectrometry (DMS) technology and multiple reaction monitoring (MRM) on a QTRAP mass spectrometer 10. Although the reproducibility performance of different lipidomics workflow and different technologies from a multi-institutional study has been well evaluated 11, none have examined the Lipidyzer performance. Recently, the performance of the targeted Lipidyzer platform has been well compared with those of the conventional untargeted LC/MS platform, and results showed that both platforms provide comparable and robust lipid profiling data from mouse plasma 12. Since, the platform was recently acquired in our lab, the primary goal of this study was to apply this new technology towards lipid profiling of mammalian tissues and evaluate its performance in this capacity, which to our knowledge has not been previously done. 3 ACS Paragon Plus Environment


Defects in lipid metabolism have been reported in metabolic syndrome, cardiovascular diseases, neurological diseases, diabetes and cancer 3. Furthermore, many individual lipid species have tissue-specific functions, for example, sphingomyelin lipids in the nerve system facilitate electrical impulse conduction 13. The human tissue proteome has been mapped using quantitative transcriptome profiling data and protein profiling data from all major organs and tissues in the human body (http://www.proteinatlas.org/). Unlike the proteome (which is genetically encoded with certain building patterns), lipid profiling in various tissues is challenging due to the non-patterned and widely varied structures and concentrations of lipids. Little work has been conducted for lipid profiling across tissues, to reveal the extent that lipids exhibit tissue specificity 14. In this study, the performance of the Lipidyzer platform based on MRM and differential ion mobility technology was evaluated, and the platform was employed to explore tissue specific lipid profiles, sex-based differences, lipid distribution in the tissues, and biological variance in the lipidome. MATERIALS and METHODS Chemicals Optima LC/MS grade methanol, dichloromethane, propanol, water and ammonium acetate (analytical grade) were purchased from Thermo Fisher Scientific (Pittsburgh, PA). A human plasma QC sample, QC spikes standards kit, isotope labeled standards for the 13 classes of lipids, SelexION tuning standard and a system suitability testing standard were purchased from SCIEX (SCIEX, MA, USA). Animal Experiment and Sample Collection C3H mice were obtained from the FDA’s National Center for Toxicological Research (NCTR) breeding colony. Animal rooms were maintained at 23°C with 50% relative humidity and a 12 h dark/12 h light cycle. The animals were raised in a pathogen free environment and had access to NIH-41 IR diet (Richmond, IN USA) and water ad libitum. Experiments were conducted in accordance with the guidelines of NCTR’s Institutional Animal Care and Use Committee (IACUC). Male and female C3H mice aged 17 and 20-21 weeks (wks) were used in this study. Animals were anaesthetized by inhalation of 0.5-2.0 % isoflurane mixed with oxygen at sacrifice. After sacrificing, the liver and heart were collected for lipidomics assays. The tissues were flash frozen and stored at -80°C prior to transferring to the analytical laboratory. 4 ACS Paragon Plus Environment

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Livers were obtained from n=3 male and n=3 female C3H mice aged 17 wks to evaluate the biological variance of the lipidome within animals; and to explore sex-based difference in lipids between the male and female mice. Heart tissues were collected from four male and one female C3H mice aged 20-21 wks and subsequently pooled. The pooled liver (from all six mice aged 17 wks including 3 male and 3 female mice), and the pooled heart (from 5 mice aged 20-21 wks including 4 male and 1 female mouse) samples were used to examine the reproducibility of the Lipidyzer platform and to evaluate the lipidome difference across tissues. Sample Processing LC/MS grade water (~250 µL, at a ratio of 10 µL/mg tissue) was added to the Eppendorf tube containing pulverized liver or heart tissue (~25 mg), followed by homogenization for 40s twice. Aliquots of 25 µL of liver homogenate (n=3/sex), 25 µL of pooled liver homogenate (n=3), 50 µL pooled heart homogenate (n=3 runs), and 50 µL QC plasma sample (n=6, SCIEX, MA, USA) were transferred to glass tubes for lipid extraction. First, all samples were diluted to 100 µL by adding the appropriate volume of water. A 25 µL aliquot of the QC spike mixture was added to each of the three QC plasma samples to make QC spike samples. Lipid extraction was achieved by using a modified version of the Bligh and Dyer extraction protocol 10, whereby 0.9 mL of H2O, 2 mL methanol, and 0.9 mL dichloromethane (DCM) was added and mixed gently, but thoroughly for 5 s. Aliquots of stable internal standard mixtures (https://sciex.com/Documents/Downloads/Certificates%20of%20Analysis/lipidyzer/IS_Kit_5040 156_LPISTDKIT_100.pdf), which contained fifty individual isotope-labeled standards that covers 13 lipid classes, were spiked into all samples. Following two rounds of extraction, the bottom layers were combined and dried under nitrogen flow and reconstituted in 0.30 mL of 10 mM ammonium acetate in 1:1 DCM/methanol and centrifuged just prior to analysis. Differential Ion Mobility-MS/MS Analysis An aliquot of 50 µL of the reconstituted sample was directly infused into a QTRAP 5500 MS with SelexION™ technology at a flow rate of 7 µL/min for targeted lipid profiling. The Lipidyzer platform contains >1000 multiple reaction monitoring (MRM) transitions in both positive and negative ionization modes to measure 13 classes of lipids including cholesterol esters (CE), ceramides (CER), dihydroceramides (DCER), diacylglycerols (DAG), free fatty acids (FFA), hexosylceramides (HCER), lactosylceramides (LCER), lysophosphatidylcholines



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(LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), phosphatidylethanolamines (PE), sphingomyelins (SM) and triacylglycerols (TAG). Individual lipid species were quantified with their appropriate internal standards using the built-in Lipidomics Workflow Manager (LWM) software, which allows for automated data acquisition, data processing and reporting. The detailed quantification processing information was well described previously12. It must be noted that most of lipid classes have multiple internal standards with varied fatty acid chain lengths and degrees of unsaturation. Twenty spectral scans were collected for each lipid, 4 outliers out of the 20 scans were discarded, the area of the16 remaining scans were averaged and used to estimate the concentration of each detected lipid. 𝐼𝐴

𝐶𝐼𝑆

The concentration was calculated as the formula below: CA = 𝐼𝐼𝑆 ∗ 𝑀𝑊𝐼𝑆 ∗ 1000; where, CA: concentration of analyte (nmol/mL), IA: intensity of the analyte, IIS: intensity of the internal standard, CIS: concentration of the internal standard (mg/mL), MWIS: molecular weight of the internal standard. The data for the detected lipids was exported to EXCEL and the concentrations reported as nmol/mL in the reconstituted solution. The software R 3.3.1 was used for statistical and multivariate analyses. Welch’s test was used to compare difference between tissues. The false discovery rate (FDR) was calculated using the Benjamini & Hockberg method. A value of p < 0.05 with FDR 6 folds of that present in the heart.

CE = cholesterol esters; CER = ceramides; DCER = dihydroceramides; DAG = diacylglycerols; FFA = free fatty acids; HCER = hexosylceramides; LCER = lactosylceramides; LPC = lysophosphatidylcholines; LPE = lysophosphatidylethanolamines; PC = phosphatidylcholines; PE = phosphatidylethanolamines; SM = sphingomyelins; and TAG = triacylglycerols.

Figure 2. Concentrations (nmol/mL) across female and male liver, pooled liver and heart per lipid class. Note: ‫ ٭‬p90%) were more abundant in the liver tissue than in the heart (Figure 5A). The class of CE lipids showed sex differences in the liver samples, although it was not determined which species was responsible for the sex difference (Fig. 2). Figure 5B showed that the most abundant CE species (CE(16:1) and CE(18:1)) had significant A

B

Figure 5. Heat map of fold changes of 787 individual lipid species across the 13 lipid classes in all individual animals (A). Fold change is log2(concentration/average for each lipid in all of the animals). Selected lipid species distribution patterns in tissues (B). Error bars denote standard deviation (SD) from n=3 replicates. sex differences (p 2-fold vs. liver. SM(20:0) and SM(20:1) were more abundant in heart (>3 fold) vs. liver. Concentrations of PE(18:0/22:6) and PE(18:1/22:6) were significantly higher (p2 folds of that in the liver. The abovementioned observations suggest that lipid species display tissue specific distribution patterns. In order to examine the lipid species profiles within a tissue, the detected concentrations of 491 TAG species, 11 LPC species, 80 PC species and 108 PE species in heart and liver are displayed in Figure 6. The liver had the highest total levels of TAG C52 containing 52 carbons 10 ACS Paragon Plus Environment

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with 0 to 8 unsaturated bonds than any other TAG species (Figure 6A), among which the most abundant TAG species is TAG52:2-FA18:1 followed by TAG52:2-FA16:0. In contrast, the heart had comparable levels of TAGs containing 50 carbons, TAGs containing 52 carbons and TAGs with 54 carbons. Interestingly, the heart had relatively higher levels of short chain TAG species (e.g. with 48 carbons) vs. liver. Furthermore, the heart had distinctly different distributions of LPCs with LPC(22:6) being the most abundant among all of the 11 LPCs (Figure 6B). The

Figure 6. Lipid profiles of the detected 491 TAG (A), 11 LPC (B), 80 PC (C) and 108 PE (D) species in heart and liver. Solid lines for heart tissue include three individual runs from the pooled heart samples; for liver includes three individual runs from the pooled liver samples, individual female (n=3) and male liver samples (n=3). The triangles at y-axis indicate the number of double bonds increasing from left to right within each group. heart had distinct unique PC species profiles with Figure 6 showing that PC(16:0/22:6) and PC(18:0/22:6) were the most abundant of all the PC species (Figure 6C). The liver had comparable levels of PC(16:0/18:2), PC(16:0/20:4), PC(16:0/22:6), PC(18:0/18:2), PC(18:0/20:4), and PC(18:0/22:6). Plots of the 108 PE species profiles, showed that the most abundant species in the heart were PE(18:0/22:6) followed by PE(16:0/22:6) and PE(18:1/22:6) (Figure 6D). In contrast, the liver contained comparable levels of the polyunsaturated PE 11 ACS Paragon Plus Environment


species, PE(16:0/22:6), PE(18:0-1/20:4) and PE(18:0-1/22:6), which is similar to the LPC species distribution in the liver. The distributions of 26 FFA, 26 CE, 12 CER, 12 HCER, 59 DAG, and 12 SM species within the liver and heart are displayed in Figure S1. No distinct distribution differences were observed in either of the tissues (Figure S1). DISCUSSION This pilot study was designed to examine the performance of the Lipidyzer platform, to examine the sex differences in lipids, and to discover tissue-specific lipid profiles. Since this pilot study only encompassed 3 mice/sex, future lipidome studies should include more animals due to a large subject biological variance present between animals. The performance of the Lipidyzer platform equipped with a QTRAP 5500 MS was evaluated. Although the CVs of all detected 787 lipid species in the QC and pooled heart and liver samples are ~10%, there are still some limitations of this lipidomics platform. The limitation includes: 1) The system can currently measure only 13 classes of lipids, which does not cover other important lipids such as acyl carnitines, phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL) and gangliosides; 2) the platform was developed to analyze human plasma for non-clinical research although based on our work it appears that lipid profiling analysis can be extended to analyze mouse liver and heart tissues as described in this report, and perhaps other mammalian tissue types. One should however, be cautious in using the absolute quantified data since the spiked-in internal standard levels should be based on the lipid levels of each animal species and specific tissues. However, relative lipid level comparisons should be reasonable and sufficient to determine significant differences in tissue lipid profile in a given tissue. Overall, the platform provides a relatively rapid and convenient means to perform broad spectrum semi-quantitative lipidomics analysis for certain lipid classes. In total, 787 lipid species from 13 lipid classes (Table S1) were detected and quantified using the spiked-in 50 stable isotope labeled lipids in the samples. Although these detected lipids represent only a portion of >100,000 lipid species in mammalian systems, results from the study can reveal the unique lipid profiles in heart and liver tissues. Jain at al. reported a similar study which measured 179 lipid species from 10 classes across 18 mouse tissues 14. However, the lipid abundance was compared based on the intensity data of the detected lipid ions, which could be biased considering the different ionization efficiency and lack of internal standards for 12 ACS Paragon Plus Environment

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quantitation 14. In order to obtain tissue atlases of lipid profiles similar to the tissue atlases of gene expression 15 and the tissue atlases of the human proteome in major organs (http://www.proteinatlas.org/), further studies are needed for lipid profiles in tissues/organs other than liver and heart. As expected, the total concentration of the TAG class was the most abundant in the liver vs. heart (Figure 2). This finding is in accordance with the fact that the liver is responsible for TAG redistribution and TAG biosynthesis from digested dietary fats or carbohydrates 16; the newly synthesized TAG is exported out of the liver to the blood. It was also observed that the total concentration of the CE class had sex differences in the liver, while no such difference was observed in the heart (Figure 2). Premenopausal women are reported to have lower incidence of hypercholesterol-related diseases (such as cardiovascular disease) than men. The observed higher levels of the total CE in the females could indicate that more CE was biosynthesized or transported into the liver from the diet for metabolic disposition. These findings are consistent with a previous report that female mice have higher levels of total hepatic CE than males 17. The two most abundant CE species, CE(18:1) and CE(16:1), contributed to sex differences (Figure 5) in the liver, and could be potential sex difference biomarkers. Sex differences observed in the study could potentially provide information for prevention, diagnosis and treatment of hypercholesterol-related diseases but further studies are needed for confirmation. PC(18:0/22:6) and PC(18:2/22:6) were more abundant than other PC species in the heart (Figure 5). This finding is consistent with the reported results that the polyunsaturated PC 40:6-8 species are relatively abundant in the heart and muscle tissue 14. Further, the PC species profiles data showed that the most abundant PCs in the heart were PC(16:0/22:6) and PC(18:0/22:6) (Figure 6). Three PCs, including PC(16:0/22:6), PC(18:0/22:6) and PC(18:2/22:6), contain one chain of docosahexaenoic acid at the C-2 position. Interestingly, LPC(22:6) containing docosahexaenoic acid was also detected as the most abundant LPC species in the heart (Figure 6). PE(18:0-1/22:6) and PE(16:0/22:6) containing docosahexaenoic acid were observed to be more abundant in the heart compared with other PE species (Figure 6). Taken together, the findings suggest that docosahexaenoic acid could be very important for heart health and/or function, although it is very challenging to define the function of the individual lipid species to the organ. Indeed, numerous epidemiological, clinical and preclinical studies 18-21 have shown that docosahexaenoic acid has beneficial effects on atherothrombotic cardiovascular disease 13 ACS Paragon Plus Environment


(CVD) and lowers the risk factors for CVD by lowering circulating TAG levels. However, no publications have reported that the heart contains higher levels of the phospholipids containing docosahexaenoic acid compared with other phospholipids. It has been reported that phospholipids containing docosahexaenoic acid are the conformation cofactors for the membrane protein complexes (e.g. rhodopsin and ion pump proteins) whose functions are related to the SR Ca2+–ATPase (SERCA) and mitochondrial respiration enzymes 22, 23. Infante et al. 24 hypothesized that high-frequency contraction muscles have higher levels of phospholipids containing docosahexaenoic acid, and their study results showed that hummingbird pectoral muscles and rattlesnake rattler muscles have higher concentrations of docosahexaenoic acid vs. other muscles with low-frequency movement in the same animal. However, no direct phospholipid analysis was conducted by Infante et al 24. To our knowledge, this is the first study to show that the heart has higher levels of phospholipids containing docosahexaenoic acid. SM(20:0) and SM(20:1) species had higher levels in the heart (Figure 5). Sphingomyelins are major component of mammalian cell membranes, and have recently been discovered to play important roles in regulation of cell growth, death, migration etc. 25. To our knowledge, no publications have reported that SM(20:0) and SM(20:1) are specific in heart tissue. SM(20:0) or SM(20:1) was not detected in plasma 26, liver 27 or other tissues 28, but were detected in the heart 29. It is unclear whether SM(20:0) and SM(20:1) are related to the heart functions. Nonetheless, the two detected sphingomyelin species could play a role in the heart function. CONCLUSION This pilot study was designed to evaluate the Lipidyzer platform performance as well as its applications in lipid profiling of various types of biological samples. This study has limitations: 1) more male and female mice need to be included in the study to better gauge the biological variations; 2) the current study was limited to examine only liver and heart tissues. In order to generate an atlas of the tissue-specific lipid distribution in mice, various tissues/organs across the body need to be quantitatively analyzed. The QC and QC spike data showed that the lipidomics data from the Lipidyzer platform was very reproducible with CV values < 10%. The PCA scores plots indicated that there were differences in lipid distribution between tissues. The total concentration of the CE lipid class, and the specific CE(16:1) and CE(18:1) species showed sex differences in the liver. The total concentration of the TAG class was the highest in the liver. 14 ACS Paragon Plus Environment

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Almost all 491 TAG species were more abundant in the liver than the heart. The heart had higher levels of docosahexaenoic acid containing phospholipids including PC(18:0/22:6), PC(18:2/22:6), LPC(22:6) PE(18:0-1/22:6) and PE(16:0/22:6), which could be related to the heart health status. SM(20:0) and SM(20:1) were also more abundant in the heart. Our results demonstrate the usefulness of the Lipidyzer platform in identifying differences in lipid profile at the tissue level and between male and female mice in specific tissues. ACKNOWLEDGEMENT and DISCLAIMER This study was supported with funds from NCTR/FDA, Jefferson, Arkansas. The opinions expressed in this manuscript do not necessarily represent those of the U.S. Food and Drug Administration. Since the Lipidyzer platform was purchased from SCIEX (SCIEX, MA, USA), we received technical support from SCIEX regarding the lipidomics method development.



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Figure Legends Figure 1. The box-plot (A) and histogram (B) of the calculated covariance of the detected 787 lipid species from female liver (F-L, n=3), male liver (M-L, n=3), pooled heart (Heart, n=3), pooled liver (Pooled-L, n=3), QC and QC spike samples. Figure 2. Concentrations (nmol/mL) across female and male liver, pooled liver and heart per lipid class. Note: ‫ ٭‬p

Evaluation of the Performance of Lipidyzer Platform and Its Application

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