A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source

Breitkopf, Susanne B.; Ricoult, Stéphane J. H.; Yuan, Min; Xu, Ying; Peake, David A.; Manning, Brendan D.; Asara, John M.

doi:10.1007/s11306-016-1157-8

Cited by 140 publications

(131 citation statements)

References 64 publications

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“…Shotgun lipidomics, i.e., direct infusion HRMS (DI-HRMS) [2] enables accurate quantification of hundreds of lipids in complex samples. Evidently, the combination of HRMS and liquid chromatography (LC) is powerful when aiming at in-depth characterization of the lipidome, as increased dynamic range improves the lipidome coverage, which in turn enables higher numbers of identified species within one analytical run [3,4]. In this work, for the first time, both HRMS and LC-HRMS have been combined to lipid class-specific fractionation as obtained by upscaling analytical supercritical fluid chromatography (SFC).…”

Section: Introductionmentioning

confidence: 99%

Preparative supercritical fluid chromatography for lipid class fractionation—a novel strategy in high-resolution mass spectrometry based lipidomics

et al. 2020

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In this work, a lipidomics workflow based on offline semi-preparative lipid class-specific fractionation by supercritical fluid chromatography (SFC) followed by high-resolution mass spectrometry was introduced. The powerful SFC approach offered separation of a wide polarity range for lipids, enabled enrichment (up to 3 orders of magnitude) of lipids, selective fractionation of 14 lipid classes/ subclasses, and increased dynamic range enabling in-depth characterization. A significantly increased coverage of low abundant lipids improving lipid identification by numbers and degree (species and molecular level) was obtained in Pichia pastoris when comparing high-resolution mass spectrometry based lipidomics with and without prior fractionation. Proof-of-principle experiments using a standard reference material (SRM 1950, NIST) for human plasma showed that the proposed strategy enabled quantitative lipidomics. Indeed, for 70 lipids, the consensus values available for this sample could be met. Thus, the novel workflow is ideally suited for lipid class-specific purification/isolation from milligram amounts of sample while not compromising on omics type of analysis (identification and quantification). Finally, compared with established fractionation/pre-concentration approaches, semi-preparative SFC is superior in terms of versatility, as it involved only volatile modifiers and salt additives facilitating any follow-up use such as qualitative or quantitate analysis or further purification down to the single lipid species level.

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Section: Introductionmentioning

confidence: 99%

Preparative supercritical fluid chromatography for lipid class fractionation—a novel strategy in high-resolution mass spectrometry based lipidomics

et al. 2020

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“…As shown in Fig 2A, each tissue and isolated adipocytes showed a similar total lipid content. A quantitative LC-MS/MS based analysis of the total lipid content extracted from BM-Ad and SC-Ad was performed using a recently developed approach that uses both positive and negative ionization modes to cover the largest spectrum of detectable lipid species (Breitkopf et al, 2017). The analysis structurally characterized and identified 818 lipid species originating from the main lipid categories that belonging to 15 lipid classes.…”

Section: Resultsmentioning

confidence: 99%

“…Lipids were resuspended with 100µl of 1:1 LC/MS grade isopropanol: acetonitrile methanol and 5 µl were injected onto the LC-MS/MS. Data acquisition was performed as previously described (Breitkopf et al, 2017). Briefly, each peaks area was calculated in both positive and negative ionization mode.…”

Section: Methodsmentioning

confidence: 99%

Yellow adipocytes comprise a new adipocyte sub-type present in human bone marrow

Attané

Estève

Chaoui

et al. 2019

Preprint

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25During energy demanding conditions, white adipocytes store triglycerides and release fatty acids through lipolysis. 26In contrast, bone marrow adipocytes (BM-Ad) increase in size during caloric restriction, suggesting this fat depot 27 exhibits precise metabolic specificity. We found subcutaneous adipocytes (SC-Ad) and BM-Ad share 28 morphological features, but possess distinct lipid metabolism. BM-Ad show enrichment in cholesterol-oriented 29 metabolism that correlates with increased free cholesterol content, while proteins involved in lipolysis were 30 downregulated. A strong down-regulation in expression of monoacylglycerol (MG) lipase was observed leading 31 to an accumulation of major MG species and accordingly the basal and induced lipolytic responses were absent in 32 BM-Ad. These features are not recapitulated in vitro using differentiated bone marrow mesenchymal stem cells. 33Since our data demonstrate that BM-Ad comprise a distinct class of adipocytes, we propose renaming them yellow 34 adipocytes. 35 36 Keywords 37Bone Marrow adipocytes / cholesterol/lipolysis / monoacylglycerol lipase / proteomic 2 and well-studied fat deposits occur in subcutaneous regions (SC-AT) and in the abdominal cavity surrounding key 3 internal organs like the pancreas and intestines (Zwick et al, 2018). Other adipose-specific deposits also form 4 around the heart, kidney, prostate in men and mammary glands in women (Zwick et al, 2018). In addition to WAT, 5 mammals also possess brown adipose tissue (BAT) located in the interscapular and supraclavicular regions, 6 representing less than 5% of the total fat mass (Leitner et al, 2017; Nedergaard et al, 2007; Saito et al, 2009; 7 Zingaretti et al, 2009). Brown adipocytes participate in non-shivering thermogenesis and possess a specific 8 morphology that includes several small lipid droplets and high mitochondria content (Bartelt & Heeren, 2014; 9 Cinti, 2001). In contrast, white adipocytes store energy as triglycerides (TG) in their unique large lipid droplet 10 (LD) after energy intake and release free fatty acids (FFA) through lipolysis in energy demanding conditions 11 (Zechner, 2015). Lipolysis occurs through a biochemical pathway that uses consecutive actions of adipose 12 triglyceride lipase (ATGL), which catalyzes the conversion of TG to diacylglycerols (DG) and hormone-sensitive 13 lipase (HSL) and hydrolyzes DAG to monoacylglycerols (MG), monoacylglycerol lipase (MAGL) and the newly 14 identified α/β hydrolase domain-containing protein 6 (ABHD6), which hydrolyzes MG to FA (Zhao et al, 2016) 15 and glycerol (Zechner, 2015). White adipocytes also have an important endocrine function as they can release 16 multiple soluble factors called adipokines, such as leptin and adiponectin (Fasshauer & Blüher, 2015). 17One intriguing adipose tissue (AT) localizes to the bone marrow called bone marrow adipose tissue (BM-AT) that 18 constitutes over 10% of the total fat mass in lean and healthy humans (Cawthorn et al, 2014). Technological 19 advances in quantitative imaging...

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“…Lipidomic analysis was performed as described previously with slight modifications (70). Briefly, total cellular lipids were extracted with methyl tert-butyl ether (MTBE) (Sigma Aldrich) from fresh cell pellets and dried in a SpeedVac concentrator (Thermo Scientific).…”

Section: Methodsmentioning

confidence: 99%

EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma

Shi

Zheng

Jiang

et al. 2020

Preprint

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29Core regulatory circuitry (CRC)-dependent transcriptional network is critical for 30 developmental tumors in children and young adults carrying few gene mutations. 31 Ewing sarcoma resembles these developmental cancers in terms of having both 87 few genomic alterations and a single epigenomic driver, we postulated that such 88 oncogenic CRC model might exist in Ewing sarcoma, to cooperate with the 89 transcriptional function of EWS-FLI1. The current study was aimed to identify 90 and characterize this CRC apparatus in Ewing sarcoma, and to elucidate its 91 functional significance in this cancer. 92 93 94 5 / 46 Results 95 96 We recently characterized the super-enhancer landscape in Ewing sarcoma and 97 confirmed the essential role of EWS-FLI1 in regulating the epigenome of this 98 cancer (11). As introduced earlier, considering that CRC is particularly important 99 for childhood developmental cancers having few genomic lesions, we postulated 100 that such CRC model might also contribute to regulating Ewing sarcoma 101 transcriptome through either cooperating with or mediating the function of EWS-102 FLI1. To test this hypothesis, we first sought to identify mathematically master 103 TFs with high inter-connectivity through binding to their super-enhancers by 104 motif scanning using our established method (18, 24, 25). Because of the central 105 role of EWS-FLI1 in establishing the enhancer landscape in Ewing sarcoma, we 106 made significant modifications of the method by requiring that all candidate TFs 107 have both EWS-FLI1 binding motif and binding peaks in their assigned super-108 enhancers. We initially focused on the A673 cell line, since it is a well-109 characterized Ewing sarcoma line with available H3K27ac and EWS-FLI1 ChIP-110 Seq results (7). As a result, a small set (n=9) of CRC candidates were identified 111 (Fig. 1A), including NKX2-2 and FOS which are known functional cooperators 112 of EWS-FLI1 (7, 26). Because CRC factors have high and specific expression in 113 their corresponding cell types, we interrogated the Cancer Cell Line Encyclopedia 114 (CCLE) dataset and noted that, compared with other 5 CRC candidates (NFATC2, 115 FOS, IRF2, ZBTB7B and MEF2D, Supplement Fig. 1), the expression of 4 116 6 / 46 candidate TFs (KLF15, NKX2-2, TCF4 and RREB1) showed restricted 117 expression pattern in Ewing sarcoma cell lines (defined as top 5 among all cell 118 types, Fig. 1B). However, it should be noted that the specificity of RREB1 119 expression was overall poor, as most cancer types had comparable mRNA levels. Identification of CRC under the regulation of EWS-FLI1 in Ewing sarcoma 120As a parallel analysis, Pearson correlation coefficient of the mRNA levels of 121 these 9 candidates was determined, considering the co-regulatory relationship 122 between CRC members. Notably, the same set of 4 factors (KLF15, NKX2-2, 123 TCF4 and RREB1) displayed strong positive correlations with each other (Fig. 124 1C). In contrast, this correlation pattern was much weaker in other non-Ewing 125 sarco...

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A relative quantitative positive/negative ion switching method for untargeted lipidomics via high resolution LC-MS/MS from any biological source

Cited by 140 publications

References 64 publications

Preparative supercritical fluid chromatography for lipid class fractionation—a novel strategy in high-resolution mass spectrometry based lipidomics

Preparative supercritical fluid chromatography for lipid class fractionation—a novel strategy in high-resolution mass spectrometry based lipidomics

Yellow adipocytes comprise a new adipocyte sub-type present in human bone marrow

EWS-FLI1 regulates and cooperates with core regulatory circuitry in Ewing sarcoma

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