Oncocytic tumors are characterized by an excessive eosinophilic, granular cytoplasm due to aberrant accumulation of mitochondria. Mutations in mitochondrial DNA occur in oncocytic thyroid tumors, but there is no information about their lipid composition, which might reveal candidate theranostic molecules. Here, we used desorption electrospray ionization mass spectrometry (DESI-MS) to image and chemically characterize the lipid composition of oncocytic thyroid tumors, as compared with nononcocytic thyroid tumors and normal thyroid samples. We identified a novel molecular signature of oncocytic tumors characterized by an abnormally high abundance and chemical diversity of cardiolipins (CL), including many oxidized species. DESI-MS imaging and IHC experiments confirmed that the spatial distribution of CLs overlapped with regions of accumulation of mitochondria-rich oncocytic cells. Fluorescent imaging and mitochondrial isolation showed that both mitochondrial accumulation and alteration in CL composition of mitochondria occurred in oncocytic tumors cells, thus contributing the aberrant molecular signatures detected. A total of 219 molecular ions, including CLs, other glycerophospholipids, fatty acids, and metabolites, were found at increased or decreased abundance in oncocytic, nononcocytic, or normal thyroid tissues. Our findings suggest new candidate targets for clinical and therapeutic use against oncocytic tumors. Cancer Res; 76(22); 6588–97. ©2016 AACR.

Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with an aberrant accumulation of mitochondria (1). Tumors composed of oncocytic cells are particularly common among thyroid neoplasms of follicular cell derivation. Clinically, oncocytic thyroid tumors (also called Hurthle cell neoplasms) have poorer oncologic outcomes than their nononcocytic counterparts and are thus considered an adverse prognostic indicator. The biological mechanisms underlying mitochondria accumulation in oncocytic tumors are not fully understood. Sequencing studies have reported upregulation of genes involved in mitochondrial biogenesis and oxidative metabolism in thyroid oncocytic tumors, including genes from the tricarboxylic acid cycle and cytosolic glycolysis (2). More recently, mutations in the mtDNA disruptive complex I have been described as potential markers of oncocytic lesions (3).

The inner mitochondrial membrane of eukaryotic cells has a complex structure and molecular composition, recognized by the presence of cardiolipins (CL), a unique class of anionic glycerophospholipid located predominantly (if not exclusively) in mitochondria. CLs have a distinctive chemical structure, composed of two phosphatidylglycerols bridged via a glycerol backbone, which displays two negative charges from the phosphate groups and four acyl chains. These complex lipids play multiple structural and functional roles in bioenergetics, mitochondrial signaling, and cellular fate pathways and are associated with individual complexes of the electron transport. Dysregulation of CL expression and composition has been increasingly investigated in biological samples using high-performance liquid chromatography mass spectrometry (HPLC-MS). Lipid analysis by HPLC-MS provides rich and quantitative information from biological samples. Yet, these time-consuming assays require sample homogenization and thus disregard cellular heterogeneity within biological tissue samples and preclude acquisition of spatial information. Recently, HPLC-MS was used to identify CLs and oxidized CL species in a rat model of traumatic brain injury (4). CLs were also identified by HPLC-MS as proliferation markers in prostate cancer cell lines, and differences in CL composition were seen in tissues from 6 prostate cancer patients, although with no statistical significance (5). In clinical research, the ratio of the intensities of CLs and monolysocardiolipins (MLCL), measured in blood by HPLC-MS, has been suggested as a screening test for Barth syndrome (6). Despite the importance of CLs in physiologic function and their role in mediating the pathology of disease states, no studies have investigated their role in human thyroid tumor tissues.

Here, we used MS imaging to chemically characterize the lipid signatures of thyroid tumors. We discovered a rich molecular signature uniquely characterized by a high abundance and diversity of CL species in oncocytic thyroid tumors. Different CL species, including oxidized CLs (ox-CL), adducts of CL, and glycerophosphocholines (PC) or diacylglycerides (DG), and MLCL were identified at high relative abundances in oncocytic tumors when compared with nononcocytic tumors and normal thyroid tissues. DESI-MS imaging and optical imaging of stained tissues, even though in a different spatial scale, confirmed that the CL distribution and highest intensity colocalized with regions of oncocytic tumor cells in thyroid tissues. Fluorescent imaging and mitochondrial isolation studies showed that mitochondrial accumulation and alteration in CL composition of mitochondria occur in oncocytic tumors cells, thus contributing the aberrant molecular signatures detected. Statistical analysis showed that CL species were increased in oncocytic tumors with the highest statistical significance, while other glycerophospholipids (GP) and fatty acids (FA) were also found to significantly discriminate oncocytic tumors, nononcocytic tumors, and normal thyroid tissues.

MS imaging has been extensively used to investigate the lipid profiles of human tumors (7, 8). In particular, ambient ionization MS imaging techniques, such as DESI-MS, allow for direct analysis of tissue sections, in the open air environment, and with minimal sample preparation (9–12). DESI-MS has been used to characterize the lipid profiles of brain (13, 14), gastric (15), breast (16, 17), and other cancers (18). Variations in the relative and total abundance of FA and most abundant complex lipids classes have been described in cancer tissues when compared with normal tissues (18). Yet, as the abundance of CL in cells and tissues is low relative to other lipids, little has been reported on the changes of CL in cancer tissue using MS imaging. Matrix-assisted laser desorption/ionization mass spectrometry has been employed to analyze CL in animal model tissues or cell lines (19–21). Using DESI-MS imaging, five different CL species were identified in MYC-induced lymphoma mice tissue (22), and a single CL species was detected in normal human gastric epithelial tissue (15). To the best of our knowledge, this is the first study to report a diverse group of CL species as molecular markers of human tumors. As lipid signatures can be readily accessed using ambient MS imaging, we expect this method to be valuable for clinical use (18).

For detailed information on materials and methods, please see Supplementary Information.

Banked human thyroid tissues

A total of 45 frozen human thyroid tissue specimens were obtained from Cooperative Human Tissue Network, Baylor College Tissue Bank, and Asterand Biosciences under approved Institutional Review Board protocol. A first set of 30 samples included 10 oncocytic thyroid tumors (8 Hurthle cell adenomas and 2 Hurthle cell carcinomas), 10 nononcocytic thyroid tumors (5 papillary thyroid carcinomas and 5 follicular thyroid adenoma), and 10 normal thyroid tissues. A second set was purchased and analyzed independently, including 5 oncocytic thyroid tumors (2 Hurthle cell adenomas and 3 Hurthle cell carcinomas) and 10 nononcocytic thyroid tumors (5 papillary thyroid carcinomas and 5 follicular thyroid carcinomas). All tissue samples were sectioned at 16-μm thick sections, and stored in a −80°C freezer until analysis.

DESI-MS imaging

A 2D Omni Spray (Prosolia Inc.) was used for tissue imaging with a spatial resolution of 150 μm. DESI-MS imaging was performed in the negative ion mode from m/z 100 to 1,500 using a hybrid LTQ-Orbitrap Elite Mass Spectrometer (Thermo Scientific). The histologically compatible solvent system dimethylformamide:acetonitrile 1:1 was used for analysis (23). For ion identification, high mass resolution/accuracy measurements and tandem MS analyses were performed in the Orbitrap and the linear ion trap using the same tissue sections analyzed.

Histopathology

The same tissue sections analyzed by DESI-MS were subjected afterwards to hematoxylin and eosin (H&E) staining. Pathologic evaluation was performed by W. Yu using light microscopy. Regions of clear diagnosis of cancer and normal thyroid tissue were assigned in the glass slides.

IHC, immunofluorescence, and confocal microscopy

For IHC, formalin-fixed tissue sections were stained for human mitochondria using primary Human Mitochondria monoclonal antibody MAB1273 (Millipore). All the H&E- and IHC-stained slides were scanned using the Aperio ScanScope imaging platform (Aperio Technologies) with a 20× objective at a spatial sampling period of 0.47 μm per pixel. Whole-slides images were viewed and analyzed by using desktop personal computers equipped with the free ScanScope software. For immunofluorescence, formalin-fixed tissues were stained using Alexa Fluor 488–conjugated anti-mitochondrial antibody MAB1273A4 (Millipore), counterstained and mounted in ProLong Gold Antifade mounting media (Thermo Fisher). Immunofluorescence images were acquired on a Zeiss LSM880 confocal microscope.

Mitochondria isolation and analysis

Mitochondria isolation was carried out following an organelle isolation protocol (24). Quantification was done using BCA Protein Assay Kit (Thermo Scientific). Concentrations were measured by comparing absorbance with standard protein calibration curve created with different concentrations of BSA. Total lipid extraction was carried out by Bligh–Dyer method.

Statistical analysis

Regions of interest in the 2D raw data obtained by DESI-MS were selected, converted to text files, and imported to R language for statistical analysis using the significance analysis of microarrays (SAM) method.

Molecular characterization of CLs in oncocytic tissue

Negative ion mode DESI-MS was used to analyze a total of 45 human thyroid samples, including 15 oncocytic thyroid tumors (10 Hurthle cell adenomas and 5 Hurthle cell carcinomas), 20 nononcocytic thyroid tumors (10 papillary thyroid carcinomas, 5 follicular carcinomas, and 5 follicular thyroid adenoma), and 10 normal thyroid tissues. The mass spectra obtained from m/z 100 to 1,500 (Supplementary Fig. S1) presented high relative abundances of several molecular ions commonly characterized as lipid species in the negative ion mode DESI mass spectra of human tissues, including FA and GP, such as glycerophosphoinositols (PI), glycerophosphoethanolamines (PE), and glycerophosphoserines (PS). Normal thyroid tissue displayed high relative abundances of PI(20:4/18:0) (m/z 885.548), PS(20:3/18:0) (m/z 812.544), PS(18:1/18:0) (m/z 788.544), PE(20:4/18:0) (m/z 766.538), PE(18:2/18:1) (m/z 742.538), and phosphatidic acids (PA) (18:1/18:0) (m/z 701.512), which are lipid ions commonly detected from mammalian tissues (Fig. 1, bottom). In contrast, the mass spectra obtained from oncocytic tumor samples showed a very distinct and reproducible profile with abnormally high relative abundances of a series of doubly charged ions in the mass range from m/z 590 to 760 and m/z 1,000 to 1,200 (Fig. 1, top). The spectra were remarkably rich in molecular diversity, and unlike what commonly observed in human cancer tissues by DESI-MS imaging. It should be noted that ions with two negative charges are characterized by m/z corresponding to half the molecular weight of the ion. Doubly charged ions are easily recognized by MS, as the 13C isotope peak is at a 0.5 m/z difference from the 12C isotope peak (Fig. 1, inset on top), whereas for singly charged ions, the 13C peak is observed with 1 m/z difference. Doubly charged lipid ions are unusually detected at high intensities from human tissues by DESI-MS analysis and stand out from other FA and GP ions, which are most commonly observed as singly charged species in the negative ion mode.

Figure 1.

Comparison of DESI-MS results for oncocytic thyroid tumor, nononcocytic thyroid tumor, and normal thyroid tissues. To aid visualization, representative negative ion mode DESI mass spectra of oncocytic tumor (top), nononcocytic tumor (middle), and normal thyroid tissue (bottom) mass spectra are shown from m/z 590 to 1,500, where CL species (red) and other GPs are detected (for full m/z range, please see Supplementary Fig. S1). Inset in top spectrum shows the mass spectrum of doubly charged CL ion in the mass region from m/z 723 to m/z 729, with a 0.5 mass difference between each peak (characteristic of doubly charged ions).

Figure 1.

Comparison of DESI-MS results for oncocytic thyroid tumor, nononcocytic thyroid tumor, and normal thyroid tissues. To aid visualization, representative negative ion mode DESI mass spectra of oncocytic tumor (top), nononcocytic tumor (middle), and normal thyroid tissue (bottom) mass spectra are shown from m/z 590 to 1,500, where CL species (red) and other GPs are detected (for full m/z range, please see Supplementary Fig. S1). Inset in top spectrum shows the mass spectrum of doubly charged CL ion in the mass region from m/z 723 to m/z 729, with a 0.5 mass difference between each peak (characteristic of doubly charged ions).

Close modal

Using high mass accuracy measurements and tandem MS analysis via collision-induced dissociation and higher energy collisional dissociation, we identified these doubly charged ions as a diverse group of CL species. Structural elucidation using lithium adducts was also explored and provided confirmatory structural information (Supplementary Fig. S2; ref. 25). CLs have been previously investigated by electrospray ionization and tandem MS and present key fragment ions that enable structural characterization (26–28). For example, tandem MS experiments of doubly charged molecular ion m/z 724.483 yielded fragment ions corresponding to 18:2-carboxylate anion (m/z 279.233), 18:1-carboxylate anion (m/z 281.249), 20:2-carboxylate anion (m/z 307.264), lyso-PA fragments (m/z 415.225, m/z 417.241, and m/z 461.249), a doubly charged ketene (m/z 593.371) arising from loss of the 18:2-fatty acyl substituent, and a fragment ion at m/z 1169.737 produced by neutral loss of FA(18:2), indicating that the molecular ions correspond to CL(20:2/18:2/18:1/16:2 or 18:2/18:2/18:2/18:1) (Supplementary Fig. S3). High mass accuracy measurements agree with the exact mass (m/z 724.4867) of proposed molecular formula (C81H144O17P2) with a mass error of −1.7 ppm. Note that isomerism of the double bonds in the FA chains of GP complicates precise structural assignment, which is why acyl chains are only tentatively assigned. Furthermore, several combinations of the four acyl chains at different positions in the CL structure are possible; thus, the exact configuration cannot be assigned by our method. In total, 31 CL molecular ions were identified and characterized, except for two CL ions that present insufficient fragment ion intensity. The singly charged CL molecular ions were also observed from m/z 1,400 to 1,500 at high relative intensities in the oncocytic tumor when compared with nononcocytic and normal thyroid tissues.

Several ox-CL species were also identified in the oncocytic tumors mass spectra. For example, tandem MS experiments of doubly charged molecular ion m/z 677.414 yielded oxidized carboxylate anion (9:1-OOH; m/z 187.099), 18:2-carboxylate anion (m/z 279.234), oxidized lyso-PA from 9:1-OOH at m/z 323.092, and lyso-PA at m/z 415.228, indicating that the CL molecular species is ox-CL(18:2/18:2/18:2/9:1(OOH); Supplementary Fig. S3; ref. 29). Altogether, 17 different ox-CLs were identified in oncocytic tumor tissues, previously unreported in human tissues. Note that these oxidized species were also observed in oncocytic tissues when no voltage was applied in the DESI source (30). Most interestingly, this oxidation effect was specific to CL, as other polyunsaturated GPs at similar relative abundances were not detected in their oxidized forms by our method. Thus, our data indicate that the detected ox-CL are endogenous molecules present in oncocytic tissues.

An uncommon series of doubly charged peaks from m/z 1,000 to 1,200 were observed in high relative abundance in oncocytic tumors when compared with nononcocytic and normal thyroid tissues. These peaks were identified using a series of tandem MS experiments as a combination of CL with DG (m/z 1,000–1,100) or, more predominantly observed, with PC (m/z 1,100–1,200), with no bridging or other additional atoms. The ion m/z 1,102.262, for example, was identified as CL + PC (106:12) using MS2 and MS3 experiments (Supplementary Fig. S3). The chemical structures of many fragment ions include structural components of both PC and CL molecules, which indicates that these species are strongly bound, through what we hypothesize to be an ionic bond within a concatenated structure. To confirm the chemical composition of these ions, we performed DESI-MS analysis on a mixture of CL and PC standards and observed the formation of these doubly charged species, which presented identical fragmentation pattern (Supplementary Fig. S4) to those observed in tissue. PCs were not observed in the negative ion mode in our experiments; thus, it is interesting to observe these molecules bound to CL species. We have not found previous reports on the observations of these doubly charged molecular ions from tissue samples.

In total, 28 CL species, 17 ox-CL, 2 MLCL, 27 CL + PC, and 27 CL + DG were identified in oncocytic tumors. The results for a representative set of these species are shown in Table 1, whereas the full list is can be found in Supplementary Table S1 (note that only C12 isotope was included for each molecule). Note that two MLCL species were identified in oncocytic thyroid tumor tissue, although at a lower relative intensity when compared with the other CL species detected. In contrast to oncocytic thyroid tumors, the mass spectra obtained from nononcocytic thyroid tumors showed high relative abundances of PI(20:4/17:0) at m/z 871.536, PI(20:4/16:0) at m/z 857.520, PI(18:2/16:0) at m/z 833.518, and PE(18:1/O-16:1) at m/z 700.530 (Fig. 1), as well as many other GPs and FAs. CL species were also observed in nononcocytic tumors but at lower relative intensities than that observed in oncocytic thyroid tumors, whereas oxidized species were either undetectable or at much lower abundance using our method. In normal thyroid tissues, although the mass spectra total ion counts were similar to those observed from tumor tissues, all ox-CL species identified in oncocytic tissues were undetectable by DESI-MS (Supplementary Fig. S5). Although the DESI-MS imaging results provide a qualitative assessment of the changes in lipid abundances, multivariate statistical analysis of the individual ions was performed to evaluate whether the observed changes are significant (results shown later in the article).

Table 1.

Representative CL species identified using high mass resolution/high mass accuracy measurements and tandem MS analyses

Measured m/zLipid classaTentative attributionExact m/zMass error (ppm)bProposed formula
592.364 MLCL CL(54:5) 592.364 0.2 C63H112O16P2 
669.414 ox-CL 20:4/18:2/16:0/9:1(OH) 669.414 −0.2 C72H126O18P2 
677.411 ox-CL 18:2/18:2/18:2/9:1(OOH) 677.411 −0.1 C72H126O19P2 
678.419 ox-CL 18:2/18:2/18:1/9:1(OOH) 678.419 −0.3 C72H128O19P2 
689.429 ox-CL 18:2/18:2/18:2/12:2(OH) 689.429 −0.2 C75H130O18P2 
690.435 ox-CL 18:2/18:2/18:1/12:2(OH) 690.437 −2.8 C75H132O18P2 
  20:4/18:1/16:0/12:2(OH)    
697.428 ox-CL CL(OO-65:8) 697.427 1.1 C75H130O19P2 
697.464 CL 18:2/18:2/18:2/14:0 697.463 0.5 C77H138O17P2 
  20:2/18:2/16:2/14:0    
701.493 CL 18:1/18:1/18:0/14:0 701.495 −0.5 C77H146O17P2 
706.487 CL 18:2/18:1/18:1/15:0 706.487 0.3 C78H144O17P2 
710.471 CL 18:2/18:2/18:2/16:1 710.471 0.1 C79H140O17P2 
723.479 CL 18:2/18:2/18:2/18:2 723.479 0.1 C81H142O17P2 
  20:4/18:2/18:2/16:0    
724.485 CL 18:2/18:2/18:2/18:1 724.487 −1.7 C81H144O17P2 
  20:2/18:2/18:1/16:2    
732.482 ox-CL 18:2/18:1/19:1/17:3(OH) 730.484 −2.8 C81H142O18P2 
  18:4(OH)/18:2/18:1/16:0    
735.478 CL 20:4/18:2/18:2/18:2 735.479 −0.7 C83H142O17P2 
736.487 CL 20:4/18:2/18:2/18:1 736.487 −0.1 C83H144O17P2 
  20:3/18:2/18:2/18:2    
737.494 CL 20:4/18:2/18:1/18:1 737.495 −0.1 C83H146O17P2 
  20:3/18:2/18:2/18:1    
  20:2/18:2/18:2/18:2    
738.502 CL 20:4/20:2/18:1/16:0 738.502 −0.2 C83H148O17P2 
  20:3/18:2/18:1/18:1    
  20:2/18:2/18:2/18:1    
745.491 ox-CL CL(O74:8) 745.491 −0.7 C83H144O18P2 
747.478 CL 22:6/20:4/18:2/16:0 747.479 −0.8 C85H142O17P2 
1,034.753 CL + DG CL + DG(108:9) 1,034.756 −2.9 C120H216O22P2 
1,035.760 CL + DG CL + DG(108:8) 1,035.764 −3.0 C120H218O22P2 
1,047.761 CL + DG CL + DG(110:10) 1,047.764 −2.0 C122H220O22P2 
1,102.259 CL + PC CL + PC(106:12) 1,102.260 −0.5 C123H222O25NP3 
1,117.282 CL + PC CL + PC(108:9) 1,117.283 −1.6 C125H228O25NP3 
Measured m/zLipid classaTentative attributionExact m/zMass error (ppm)bProposed formula
592.364 MLCL CL(54:5) 592.364 0.2 C63H112O16P2 
669.414 ox-CL 20:4/18:2/16:0/9:1(OH) 669.414 −0.2 C72H126O18P2 
677.411 ox-CL 18:2/18:2/18:2/9:1(OOH) 677.411 −0.1 C72H126O19P2 
678.419 ox-CL 18:2/18:2/18:1/9:1(OOH) 678.419 −0.3 C72H128O19P2 
689.429 ox-CL 18:2/18:2/18:2/12:2(OH) 689.429 −0.2 C75H130O18P2 
690.435 ox-CL 18:2/18:2/18:1/12:2(OH) 690.437 −2.8 C75H132O18P2 
  20:4/18:1/16:0/12:2(OH)    
697.428 ox-CL CL(OO-65:8) 697.427 1.1 C75H130O19P2 
697.464 CL 18:2/18:2/18:2/14:0 697.463 0.5 C77H138O17P2 
  20:2/18:2/16:2/14:0    
701.493 CL 18:1/18:1/18:0/14:0 701.495 −0.5 C77H146O17P2 
706.487 CL 18:2/18:1/18:1/15:0 706.487 0.3 C78H144O17P2 
710.471 CL 18:2/18:2/18:2/16:1 710.471 0.1 C79H140O17P2 
723.479 CL 18:2/18:2/18:2/18:2 723.479 0.1 C81H142O17P2 
  20:4/18:2/18:2/16:0    
724.485 CL 18:2/18:2/18:2/18:1 724.487 −1.7 C81H144O17P2 
  20:2/18:2/18:1/16:2    
732.482 ox-CL 18:2/18:1/19:1/17:3(OH) 730.484 −2.8 C81H142O18P2 
  18:4(OH)/18:2/18:1/16:0    
735.478 CL 20:4/18:2/18:2/18:2 735.479 −0.7 C83H142O17P2 
736.487 CL 20:4/18:2/18:2/18:1 736.487 −0.1 C83H144O17P2 
  20:3/18:2/18:2/18:2    
737.494 CL 20:4/18:2/18:1/18:1 737.495 −0.1 C83H146O17P2 
  20:3/18:2/18:2/18:1    
  20:2/18:2/18:2/18:2    
738.502 CL 20:4/20:2/18:1/16:0 738.502 −0.2 C83H148O17P2 
  20:3/18:2/18:1/18:1    
  20:2/18:2/18:2/18:1    
745.491 ox-CL CL(O74:8) 745.491 −0.7 C83H144O18P2 
747.478 CL 22:6/20:4/18:2/16:0 747.479 −0.8 C85H142O17P2 
1,034.753 CL + DG CL + DG(108:9) 1,034.756 −2.9 C120H216O22P2 
1,035.760 CL + DG CL + DG(108:8) 1,035.764 −3.0 C120H218O22P2 
1,047.761 CL + DG CL + DG(110:10) 1,047.764 −2.0 C122H220O22P2 
1,102.259 CL + PC CL + PC(106:12) 1,102.260 −0.5 C123H222O25NP3 
1,117.282 CL + PC CL + PC(108:9) 1,117.283 −1.6 C125H228O25NP3 

aCL = cardiolipin (X:Y) denotes the total number of carbons and double bonds in the fatty acid chains.

bMass errors were calculated on the basis of the exact monoisotopic m/z of the deprotonated form of the assigned molecules.

CL distribution correlates with oncocytic cells and mitochondria accumulation in tissues

2D DESI-MS experiments were performed to examine the spatial distribution of the molecular ions detected from thyroid tissues (Fig. 2A). In particular, we were interested to investigate whether the spatial distribution of CL ions colocalized with specific histologic features in oncocytic tumor tissues. Figure 2B shows the DESI-MS images obtained for selected molecular ions for an oncocytic sample, a nononcocytic sample, and a normal thyroid tissue sample (additional imaging results are shown in Supplementary Fig. S6). Optical images of the same tissue section that were H&E stained after DESI-MS are also presented (23). On a microscopic scale, all oncocytic tumors analyzed showed characteristic histologic features with enlarged cells of high cytoplasmic volume that accommodates the increased number of mitochondria. In many samples, regions predominantly composed of cancer cells were observed adjacent or within regions defined as regions of fibrosis tissues. These tissue regions (hundreds of micrometers) were spatially assigned by pathologic evaluation and were discernable in the DESI-MS images (spatial resolution of 150 μm). In oncocytic tumors, the molecular distribution of CL species was colocalized, homogeneous, and remarkably high within the regions of oncocytic tumor cells and was in lower intensities in fibrosis regions in sample, as shown for m/z 738.502 and m/z 723.479 (Fig. 2B). Similar spatial distribution was observed for other CL, ox-CL, CL + PC, and CL + DG molecular ions (Fig. 3). In particular, all ox-CL showed highly similar spatial distributions with increased relative intensities observed in regions with accumulation of oncocytic thyroid tumor cells and absence in adjacent normal cells or fibrosis. In all normal thyroid tissue analyzed, common patterns of the cellular organization were observed, with spherical colloid surrounded by a single layer of follicular cells and scattered parafollicular cells. The molecular images obtained for normal tissues showed lipid signal colocalized with follicular cells (Fig. 2B). Colloid did not show lipid profiles and are thus seen as dark regions in the DESI-MS ion images. Nononcocytic follicular and papillary thyroid carcinoma showed typical histologic patterns and displayed a homogenous molecular distribution of the most abundant molecular ions within regions of tumor cell.

Figure 2.

Experimental design for tissue analysis and imaging using DESI-MS. A, workflow for imaging and mitochondria isolation experiments in thyroid tissues. N, normal thyroid tissues; NO, nononcocytic tumor; O, oncocytic tumor. B, DESI-MS analysis of an oncocytic tumor, nononcocytic tumor, and normal thyroid tissues. The optical images on the left are from the same tissue sections that were H&E stained after analysis by nondestructive DESI-MS imaging. Scale bar, 4 mm. Six representative ion images from different lipid ions, including PI(20:4/18:0) at m/z 885.548, PS(18:1/18:0) at m/z 788.544, PE(20:4/18:0) at m/z 766.538, CL(20:4/20:2/18:1/16:0 or 20:3/18:2/18:1/18:1 or 20:2/18:2/18:2/18:1) at m/z 738.502, CL(18:2/18:2/18:2/18:2 or 20:4/18:2/18:2/16:0) at m/z 723.479, and FA(20:4) at m/z 303.233, are represented.

Figure 2.

Experimental design for tissue analysis and imaging using DESI-MS. A, workflow for imaging and mitochondria isolation experiments in thyroid tissues. N, normal thyroid tissues; NO, nononcocytic tumor; O, oncocytic tumor. B, DESI-MS analysis of an oncocytic tumor, nononcocytic tumor, and normal thyroid tissues. The optical images on the left are from the same tissue sections that were H&E stained after analysis by nondestructive DESI-MS imaging. Scale bar, 4 mm. Six representative ion images from different lipid ions, including PI(20:4/18:0) at m/z 885.548, PS(18:1/18:0) at m/z 788.544, PE(20:4/18:0) at m/z 766.538, CL(20:4/20:2/18:1/16:0 or 20:3/18:2/18:1/18:1 or 20:2/18:2/18:2/18:1) at m/z 738.502, CL(18:2/18:2/18:2/18:2 or 20:4/18:2/18:2/16:0) at m/z 723.479, and FA(20:4) at m/z 303.233, are represented.

Close modal
Figure 3.

ox-CL, MLCL, and CL + PC and CL + DG ions are observed in regions of tissue samples with accumulation of oncocytic cells. DESI-MS images display the spatial distribution of selected CL species in an oncocytic thyroid tumor sample, as well as the optical image of the H&E-stained tissue section.

Figure 3.

ox-CL, MLCL, and CL + PC and CL + DG ions are observed in regions of tissue samples with accumulation of oncocytic cells. DESI-MS images display the spatial distribution of selected CL species in an oncocytic thyroid tumor sample, as well as the optical image of the H&E-stained tissue section.

Close modal

To evaluate whether CL distribution correlates with regions of mitochondrial accumulation in oncocytic tumors, we performed IHC with an anti-mitochondrial antibody in tissues sections adjacent to those imaged by DESI-MS. Positive staining for mitochondria was observed for all thyroid tumors analyzed, whereas negative (weak) staining was observed for all normal thyroid tissues. As expected, strong mitochondrial staining are seen in all oncocytic tissues (Supplementary Fig. S7). Spatial agreement was observed between regions of strong mitochondrial staining in oncocytic tissues and regions of high relative intensities of CL species in DESI-MS images. Papillary thyroid carcinoma that presented higher relative abundances of CL also showed mitochondria staining by IHC.

Mitochondria accumulates in oncocytic thyroid tumor cells

To further investigate the mitochondrial distribution within the cells of thyroid tissues, we performed immunofluorescence staining with an anti-mitochondrial antibody (green) and nuclear DAPI staining (blue) in adjacent sections of thyroid tissues (Fig. 4A). Confocal microscopy images obtained for oncocytic tumors show high density staining of mitochondria. A punctate staining pattern showcases accumulation of mitochondria within the cellular cytoplasm in oncocytic tumors. Nononcocytic follicular carcinoma shows less pronounced accumulation of mitochondria in scattered cells, lower than that observed for oncocytic tumors, and significantly higher than normal tissue. To provide a quantitative assessment of mitochondrial in thyroid tissue, tissues were homogenized and mitochondria was isolated following a specific organelle isolation protocol (Fig. 2A; ref. 24). Quantification of the total protein content in the isolated mitochondria pallet by BCA assay shows that there is on average 2.5 times more protein present in the mitochondrial fraction of oncocytic tumor tissues when compared with normal thyroid tissues. Normal thyroid samples contained on average 8.8 mg of protein/g of tissue sample, whereas oncocytic tumor samples contained 22.2 mg of protein/g of tissue sample and nononcocytic thyroid tumors contained 15.6 mg of protein/g of tissue sample (Fig. 4B). The changes in mitochondrial protein content between groups were found to be statistically significantly (P < 0.001 using a one-way ANOVA test).

Figure 4.

Mitochondria accumulation and changes in mitochondrial CL composition occur in oncocytic tumors. A, confocal images for oncocytic tumor (O1 and O2), nononcocytic tumor (NO1 and NO2), and normal (N1 and N2) thyroid tissues stained with anti-mitochondrial antibody (green) and DAPI nuclear staining (blue). B, quantification of the total protein content of isolated mitochondria pallet obtained from normal thyroid tissues (N), nononcocytic thyroid tumors (NO), and oncocytic thyroid tumors (O; *, P < 0.001). C, normalized CL intensities of the isolated mitochondria pallets obtained from normal thyroid tissues (N), nononcocytic thyroid tumors (NO), and oncocytic thyroid tumors (O) at the same concentration (3 μg protein/g of tissue; **, P < 0.001). P < 0.001 was considered as significant.

Figure 4.

Mitochondria accumulation and changes in mitochondrial CL composition occur in oncocytic tumors. A, confocal images for oncocytic tumor (O1 and O2), nononcocytic tumor (NO1 and NO2), and normal (N1 and N2) thyroid tissues stained with anti-mitochondrial antibody (green) and DAPI nuclear staining (blue). B, quantification of the total protein content of isolated mitochondria pallet obtained from normal thyroid tissues (N), nononcocytic thyroid tumors (NO), and oncocytic thyroid tumors (O; *, P < 0.001). C, normalized CL intensities of the isolated mitochondria pallets obtained from normal thyroid tissues (N), nononcocytic thyroid tumors (NO), and oncocytic thyroid tumors (O) at the same concentration (3 μg protein/g of tissue; **, P < 0.001). P < 0.001 was considered as significant.

Close modal

Alteration in CL composition occurs in the mitochondria of oncocytic human tumor cells

To investigate whether the abnormally high relative abundance and diversity of CL species detected from oncocytic tissues were solely related to the accumulation of mitochondria per oncocytic cell or were also associated to an alteration in the CL composition of the mitochondria membrane, we diluted the isolated mitochondrial pallets to the same concentration (3 mg protein/g of tissue) for all tissues, performed a lipid extraction, and analyzed them using the same conditions used for DESI-MS imaging of tissue sections. The mass spectra obtained showed a higher relative intensity of CL species from the mitochondria isolated from oncocytic tumors when compared with nononcocytic tumors and normal thyroid tissues (Supplementary Fig. S8). To compare the CL abundance within the samples, we normalized the total ion counts of CL species to the total lipid counts in the spectra obtained from isolated mitochondria. The average normalized value was 0.081 for oncocytic tumors, 0.037 for nononcocytic tumors, and 0.002 for normal tissue (Fig. 4C), which allows discrimination between these groups with statistical significance (P < 0.001 using a one-way ANOVA test). These results confirm that besides mitochondria accumulation, an alteration in the CL composition of the mitochondrial membrane occurs in oncocytic thyroid cells. These biological phenomena collectively contribute to the abnormally high relative intensities and diversity of CL species detected directly from oncocytic tumor tissue in our DESI-MS imaging experiments.

Lipids are molecular markers of oncocytic tumors

To evaluate whether the changes in relative abundance of the molecular ions observed in DESI mass spectra and images obtained were statistically significant, we applied SAM statistical analysis to our complex DESI-MS imaging dataset. Mass spectral data were extracted from regions of interest of a single predominant histologic composition for the first set of 30 samples investigated (i.e., cancer cells or normal follicular cells). SAM identifies whether the change in the abundance of a molecular ion (m/z value) is statistically significant between the three different phenotypes by computing a contrast value that measures the average change in the peak intensity for that m/z between the groups (31). Repeated permutations were used to determine whether the change is significantly related to the phenotype and to estimate the percentage of molecular ions identified by chance, the FDR. The mean intensity value for all samples for a certain m/z was set to zero, so that the contrast values obtained represent the mean fold increase (positive contrast) or decrease (negative contrast) for the groups when compared with the overall mean intensity value. From all the ions detected (m/z 100–1,500) for all the samples analyzed, 219 different molecular ions were selected with FDR <5%. As expected, ions corresponding to MLCL, ox-CL, CLs, CL + PC, and CL + DG presented the most significant changes in average abundances between the three groups by SAM analysis. For example, the singly charged CL(20:4/18:2/18:2/16:0 or 18:2/18:2/18:2/18:2) detected at m/z 1,447.975 presented the highest contrast values of −1.927 for normal tissue, −0.845 for nononcocytic tumors, and +2.772 for oncocytic tumors (FDR = 0). Figure 5A shows the overall trend in contrast values obtained for the CL species selected by SAM (FDR < 5%). As observed, all CL species present positive values for oncocytic tumors, which demonstrates that these lipids are significant for discriminating oncocytic tumors from nononcocytic tumors and normal thyroid tissues. Box plots for selected ions are shown in Fig. 5B. Highly reproducible results that corroborate these findings were obtained in a set of 15 independent thyroid tumors analyzed (Supplementary Fig. S9). The remaining GPs selected by SAM (FDR < 5%), including PI, PE, and PG, presented no clear trends in contrast values within the three groups.

Figure 5.

SAM analysis for identifying statistically significant ions (m/z) from DESI-MS data. A, comparison of contrast values of CLs among oncocytic thyroid tumors, nononcocytic thyroid tumors, and normal thyroid tissue (n = 30). The “contrast value” is the average normalized intensity for that group relative to the overall average intensity. B, box plots show the results obtained using SAM for selected ions, including MLCL, ox-CL, CLs, and CL+PC species, with FDR = 0.

Figure 5.

SAM analysis for identifying statistically significant ions (m/z) from DESI-MS data. A, comparison of contrast values of CLs among oncocytic thyroid tumors, nononcocytic thyroid tumors, and normal thyroid tissue (n = 30). The “contrast value” is the average normalized intensity for that group relative to the overall average intensity. B, box plots show the results obtained using SAM for selected ions, including MLCL, ox-CL, CLs, and CL+PC species, with FDR = 0.

Close modal

Accumulation of CL-rich mitochondria is a fundamental characteristic of oncocytic tumors. Mutations in mitochondrial DNA have been previously described in oncocytic thyroid tumors, yet, little is known on their lipid composition. In this study, we used DESI-MS to image and chemically characterize the lipid composition of thyroid tumors. We discovered a novel molecular signature in oncocytic tumors characterized by an abnormally high abundance and chemical diversity of CL species. DESI-MS imaging and IHC experiments confirmed that the spatial distribution of these molecular ions overlapped with regions of accumulation of mitochondria-rich oncocytic cells. Fluorescence imaging confirmed that the oncocytic tumors investigated presented high accumulation of mitochondria when compared with nononcocytic and normal thyroid tissue.

Using high mass accuracy, high mass resolution, and tandem MS experiments, we identified 101 different CL-containing molecular ions directly from oncocytic thyroid tissues. MS imaging of this large amount and diversity of CL species is unprecedented in untreated human tissues. Among these, two MLCL, which are intermediate molecules in CL remodeling (32), were detected in oncocytic thyroid tumor tissues. In addition, 54 intriguing doubly charged molecular ions composed of CL bound to PCs or DGs were seen at high relative abundances in oncocytic tumors when compared with nononcocytic or normal thyroid tissues. The mitochondrial inner membrane of eukaryotic cells has a unique composition of GPs, predominantly composed of PCs, CLs, and PEs, although the exact percent composition of lipids in human mitochondria is not known (33). Thus, the detection of CL + PC molecular ions, although unexpected, is not surprising considering the composition and spatial proximity of these molecules in the inner mitochondria membrane of oncocytic tumors.

Remarkably, we identified 17 different ox-CLs in oncocytic tumors. Oxidization of other abundant polyunsaturated phospholipids was not observed in our experiments, which indicates that this phenomenon is primarily occurring for CL, likely due to mitochondria dysregulation, which is known to occur in oncocytic thyroid tumors (2). CL oxidation has been implicated in degenerative diseases and reported in neuronal tissue that has suffered traumatic brain injury (4). Oxidative stress has been largely connected to tumorigenesis. In the mitochondrial inner membrane, CLs are found in association with the components of the electron transport chain (ETC), which generates reactive oxygen species. The proximity of the FA chains of CL to the various ETC complexes makes it a likely target of oxidative damage (29). Furthermore, redistribution of mitochondrial membrane CLs and accumulation of CL oxidation products through interactions with cytochrome c are required stages in the cellular apoptotic program, a process known to occur in tumor progression (34). Recently, ox-CLs were detected in PC-3 prostate cancer cell lines, although their content did not correlate with the proliferation of cells (5). We have also detected ox-CLs in human kidney oncocytic tumors (unpublished), further suggesting their importance in oncocytic neoplasms, although more rigorous studies are needed to determine their biological role in human tumors.

This is the first study to report an abnormal expression and composition of lipids, such as CLs, in human thyroid tumor tissues. Through immunofluorescence imaging, DESI-MS imaging, and mitochondria isolation experiments, we demonstrated that this aberrant CL signature is related to: (i) accumulation of mitochondria in oncocytic tumors; and (ii) dysregulation (increased abundance and diversified structures) of CL composition in mitochondria membrane of oncocytic tumor cells. Gene expression profiling studies have suggested a profound modification of energy metabolism in oncocytic tumors (2). Genes coding for subunits of the respiratory chain enzymes, glycolytic enzymes, and energy metabolism enzymes involved in glycolysis, the tricarboxylic acid cycle are overexpressed in oncocytic tumors, many of which are directly involved in lipid biogenesis and metabolism. Our results further suggest that dysregulation of mitochondria and lipid metabolism are relevant in oncocytic tumors and provide novel molecular information for deciphering the biological mechanisms involved in these tumors.

The thyroid is the main hormonal regulator of lipid biogenesis and mitochondrial function (35). Nevertheless, no previous studies have investigated the lipid signatures of thyroid tumors. We show that lipids are molecular markers of oncocytic tumors with statistical significance. Although direct MS imaging does not provide a quantitative assessment of molecules in tissues, using SAM, 219 distinct molecular ions (FDR < 5%), including various lipids and metabolites, were found at increased or decreased relative abundance in oncocytic, nononcocytic, or normal thyroid tissues. Besides CL, significant changes in FA abundances were also observed using statistical analysis. This rich lipid signature is characteristic and diagnostic of oncocytic phenotypes. Although our sample size is not sufficient for discriminating adenomas and carcinomas within the oncocytic tumor group, our pilot study gives further rationale to explore this problem using the molecular information obtained by ambient ionization MS. Importantly, these unprecedented findings provide possibilities for new therapeutic targets for oncocytic tumors.

As lipid signatures can be readily accessed from tissue samples using ambient ionization MS, we expect this method to be valuable for diagnosis of thyroid cancers and clinical use (18). Nondestructive DESI-MS can be adapted for fine needle aspiration biopsy analysis, the gold-standard method for preoperative diagnosis of thyroid lesions. With further increase in sample size and analysis of different tumor types, we expect to identify unique molecular signatures in various types of thyroid neoplasia to enhance diagnosis of nodules (especially those deemed indeterminate) and thus overcome current limitations of thyroid cytology.

Our work showcases the power of ambient ionization MS for CL imaging in biological tissues and is relevant to a variety of applications. Dysregulation of mitochondria occurs in many pathologies besides cancer. Lipids and their oxidized counterparts have been increasingly appreciated as important molecular markers and investigated to uncover biological pathways in disease (36–38). Further studies will be performed to extensively investigate alterations in small metabolites, FAs, CLs, and other GPs in thyroid tumors.

J. Zhang and L.S. Eberlin have ownership interest (including patents) in a patent. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J. Zhang, J. Suliburk, L.S. Eberlin

Development of methodology: J. Zhang, J. Suliburk, L.S. Eberlin

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Zhang, W. Yu, S.W. Ryu, J. Lin, G. Buentello, J. Suliburk, L.S. Eberlin

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Zhang, S.W. Ryu, R. Tibshirani, L.S. Eberlin

Writing, review, and/or revision of the manuscript: J. Zhang, W. Yu, J. Suliburk, L.S. Eberlin

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Zhang, G. Buentello, J. Suliburk, L.S. Eberlin

Study supervision: L.S. Eberlin

We thank Clara Feider, Marta Sans Escofet, Josh Bryant, Dean Appling, Emily L. Que, and Honggang Nie for assistance with experiments and valuable discussions. This study made use of the Research Histology, Pathology, and Imaging core supported by P30 CA16672 DHHS/NCI Cancer Center Support Grant. We also thank Collene Jeter (Flow Cytometry and Cell Imaging Core, MD Anderson Cancer Center) for performing immunofluorescence experiments. Tissue samples were provided by the Baylor College of Medicine Tissue Bank and the Cooperative Human Tissue Network, which is funded by the NCI.

This work was supported by the NIH/NCI through grant 4R00CA190783-02.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Tallini
G
. 
Oncocytic tumours
.
Virchows Arch
1998
;
433
:
5
12
.
2.
Baris
O
,
Savagner
F
,
Nasser
V
,
Loriod
B
,
Granjeaud
S
,
Guyetant
S
, et al
Transcriptional profiling reveals coordinated up-regulation of oxidative metabolism genes in thyroid oncocytic tumors
.
J Clin Endocrinol Metab
2004
;
89
:
994
1005
.
3.
Gasparre
G
,
Porcelli
AM
,
Bonora
E
,
Pennisi
LF
,
Toller
M
,
Iommarini
L
, et al
Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors
.
Proc Natl Acad Sci U S A
2007
;
104
:
9001
06
.
4.
Ji
J
,
Kline
AE
,
Amoscato
A
,
Samhan-Arias
AK
,
Sparvero
LJ
,
Tyurin
VA
, et al
Lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of brain injury
.
Nat Neurosci
2012
;
15
:
1407
13
.
5.
Sapandowski
A
,
Stope
M
,
Evert
K
,
Evert
M
,
Zimmermann
U
,
Peter
D
, et al
Cardiolipin composition correlates with prostate cancer cell proliferation
.
Mol Cell Biochem
2015
;
410
:
175
85
.
6.
Corcelli
A
,
Angelini
R
,
Lobasso
S
,
Bowron
A
,
Steward
C
. 
Monolysocardiolipin/cardiolipin ratio of intact leukocytes as novel tool for the screening of Barth Syndrome
.
FASEB J
2015
;
29
.
7.
Norris
JL
,
Caprioli
RM
. 
Analysis of tissue specimens by matrix-assisted laser desorption/ionization imaging mass spectrometry in biological and clinical research
.
Chem Rev
2013
;
113
:
2309
42
.
8.
Wu
C
,
Dill
AL
,
Eberlin
LS
,
Cooks
RG
,
Ifa
DR
. 
Mass spectrometry imaging under ambient conditions
.
Mass Spectrom Rev
2013
;
32
:
218
43
.
9.
Wiseman
JM
,
Ifa
DR
,
Song
QY
,
Cooks
RG
. 
Tissue imaging at atmospheric pressure using desorption electrospray ionization (DESI) mass spectrometry
.
Angew Chem-Int Ed
2006
;
45
:
7188
92
.
10.
Takats
Z
,
Wiseman
JM
,
Gologan
B
,
Cooks
RG
. 
Mass spectrometry sampling under ambient conditions with desorption electrospray ionization
.
Science
2004
;
306
:
471
73
.
11.
Hsu
C-C
,
Dorrestein
PC
. 
Visualizing life with ambient mass spectrometry
.
Curr Opin Biotechnol
2015
;
31
:
24
34
.
12.
Nemes
P
,
Vertes
A
. 
Ambient mass spectrometry for in vivo local analysis and in situ molecular tissue imaging
.
TrAC Trends Anal Chem
2012
;
34
:
22
34
.
13.
Jarmusch
AK
,
Pirro
V
,
Baird
Z
,
Hattab
EM
,
Cohen-Gadol
AA
,
Cooks
RG
. 
Lipid and metabolite profiles of human brain tumors by desorption electrospray ionization-MS
.
Proc Natl Acad Sci U S A
2016
;
113
:
1486
91
.
14.
Eberlin
LS
,
Norton
I
,
Dill
AL
,
Golby
AJ
,
Ligon
KL
,
Santagata
S
, et al
Classifying human brain tumors by lipid imaging with mass spectrometry
.
Cancer Res
2012
;
72
:
645
54
.
15.
Eberlin
LS
,
Tibshirani
RJ
,
Zhang
JL
,
Longacre
TA
,
Berry
GJ
,
Bingham
DB
, et al
Molecular assessment of surgical-resection margins of gastric cancer by mass-spectrometric imaging
.
Proc Natl Acad Sci U S A
2014
;
111
:
2436
41
.
16.
Calligaris
D
,
Caragacianu
D
,
Liu
X
,
Norton
I
,
Thompson
CJ
,
Richardson
AL
, et al
Application of desorption electrospray ionization mass spectrometry imaging in breast cancer margin analysis
.
Proc Natl Acad Sci U S A
2014
;
111
:
15184
89
.
17.
Guenther
S
,
Muirhead
LJ
,
Speller
AVM
,
Golf
O
,
Strittmatter
N
,
Ramakrishnan
R
, et al
Spatially resolved metabolic phenotyping of breast cancer by desorption electrospray ionization mass spectrometry
.
Cancer Res
2015
;
75
:
1828
37
.
18.
Ifa
DR
,
Eberlin
LS
. 
Ambient ionization mass spectrometry for cancer diagnosis and surgical margin evaluation
.
Clin Chem
2016
;
62
:
111
23
.
19.
Amoscato
AA
,
Sparvero
LJ
,
He
RR
,
Watkins
S
,
Bayir
H
,
Kagan
VE
. 
Imaging mass spectrometry of diversified cardiolipin molecular species in the brain
.
Anal Chem
2014
;
86
:
6587
95
.
20.
Angelini
R
,
Lobasso
S
,
Gorgoglione
R
,
Bowron
A
,
Steward
CG
,
Corcelli
A
. 
Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome
.
J Lipid Res
2015
;
56
:
1787
94
.
21.
Sparvero
LJ
,
Amoscato
AA
,
Kochanek
PM
,
Pitt
BR
,
Kagan
VE
,
Bayir
H
. 
Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury
.
J Neurochem
2010
;
115
:
1322
36
.
22.
Eberlin
LS
,
Gabay
M
,
Fan
AC
,
Gouw
AM
,
Tibshirani
RJ
,
Felsher
DW
, et al
Alteration of the lipid profile in lymphomas induced by MYC overexpression
.
Proc Natl Acad Sci U S A
2014
;
111
:
10450
55
.
23.
Eberlin
LS
,
Ferreira
CR
,
Dill
AL
,
Ifa
DR
,
Cheng
L
,
Cooks
RG
. 
Non-destructive, histologically compatible tissue imaging by desorption electrospray ionization mass spectrometry
.
Chembiochem
2011
;
12
:
2129
32
.
24.
Frezza
C
,
Cipolat
S
,
Scorrano
L
. 
Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts
.
Nat Protoc
2007
;
2
:
287
95
.
25.
Hsu
FF
,
Bohrer
A
,
Turk
J
. 
Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry
.
J Am Soc Mass Spectrom
1998
;
9
:
516
26
.
26.
Hsu
FF
,
Turk
J
,
Rhoades
ER
,
Russell
DG
,
Shi
YX
,
Groisman
EA
. 
Structural characterization of cardiolipin by tandem quadrupole and multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization
.
J Am Soc Mass Spectrom
2005
;
16
:
491
504
.
27.
Han
XL
,
Yang
K
,
Yang
JY
,
Cheng
H
,
Gross
RW
. 
Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples
.
J Lipid Res
2006
;
47
:
864
79
.
28.
Kiebish
MA
,
Han
XL
,
Cheng
H
,
Chuang
JH
,
Seyfried
TN
. 
Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer
.
J Lipid Res
2008
;
49
:
2545
56
.
29.
Kim
J
,
Minkler
PE
,
Salomon
RG
,
Anderson
VE
,
Hoppel
CL
. 
Cardiolipin: characterization of distinct oxidized molecular species
.
J Lipid Res
2011
;
52
:
125
35
.
30.
Pasilis
SP
,
Kertesz
V
,
Van Berkel
GJ
. 
Unexpected analyte oxidation during desorption electrospray ionization-mass spectrometry
.
Anal Chem
2008
;
80
:
1208
14
.
31.
Storey
JD
,
Tibshirani
R
. 
Statistical significance for genomewide studies
.
Proc Natl Acad Sci U S A
2003
;
100
:
9440
45
.
32.
Tyurina
YY
,
Poloyac
SM
,
Tyurin
VA
,
Kapralov
AA
,
Jiang
JF
,
Anthonymuthu
TS
, et al
A mitochondrial pathway for biosynthesis of lipid mediators
.
Nat Chem
2014
;
6
:
542
52
.
33.
Gohil
VM
,
Greenberg
ML
. 
Mitochondrial membrane biogenesis: phospholipids and proteins go hand in hand
.
J Cell Biol
2009
;
184
:
468
72
.
34.
Kagan
VE
,
Tyurin
VA
,
Jiang
JF
,
Tyurina
YY
,
Ritov
VB
,
Amoscato
AA
, et al
Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors
.
Nat Chem Biol
2005
;
1
:
223
32
.
35.
Hoch
FL
. 
Lipids and thyroid-hormones
.
Prog Lipid Res
1988
;
27
:
199
270
.
36.
Camarda
R
,
Zhou
AY
,
Kohnz
RA
,
Balakrishnan
S
,
Mahieu
C
,
Anderton
B
, et al
Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer
.
Nat Med
2016
;
22
:
427
32
.
37.
Gross
RW
,
Han
X
. 
Lipidomics at the interface of structure and function in systems biology
.
Chem Biol
2011
;
18
:
284
91
.
38.
Buas
MF
,
Gu
HW
,
Djukovic
D
,
Zhu
JJ
,
Drescher
CW
,
Urban
N
, et al
Identification of novel candidate plasma metabolite biomarkers for distinguishing serous ovarian carcinoma and benign serous ovarian tumors
.
Gynecol Oncol
2016
;
140
:
138
44
.