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Development and use of advanced mass spectrometry techniques for the characterization of cellular and mitochondrial lipidomic profiling in control fibroblasts and Parkinson's disease patients

C.D. Calvano

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

Interdipartimental Center SMART, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

E-mail : aa

A.M. Sardanelli

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari "Aldo Moro", P.zza G. Cesare, 11,70124 Bari, Italy

Department of Medicine, Campus Bio-Medico University of Rome, Via A. del Portillo 21, 00128 Roma, Italy

G. Ventura

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

M. Glaciale

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

L. Savino

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari "Aldo Moro", P.zza G. Cesare, 11,70124 Bari, Italy

I. Losito

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

Interdipartimental Center SMART, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

F.Palmisano

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

Interdipartimental Center SMART, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

T.R.I. Cataldi

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

Interdipartimental Center SMART, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy

DOI: 10.15761/TPBA.1000102

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Abstract

Lipidomics (a lipid-targeted metabolomics) aims at global analysis of lipids in biological systems. Recently, lipidomics research has received increased attention due to the well- recognized role of lipids in numerous human diseases. For instance, altered lipid pathways in the primary visual cortex and the anterior cingulate have been demonstrated in Parkinson’s disease (PD) by analyzing post-mortem tissues from patients in advanced neuronal degeneration stage. Such an approach, however, hinders the identification of the first neuronal changes. Skin fibroblasts have been recently proposed as a useful model of primary human cells, capable of reflecting the chronological and biological aging of the patients, according to their polygenic predisposition and environmental etiopathology. Here, hydrophilic interaction liquid chromatography coupled to electrospray ionization-Fourier- transform mass spectrometry was developed to characterize polar lipids occurring in human skin fibroblasts. Different lipid extraction protocols were tested, and Bligh Dyer strategy was selected as the most informative in terms of lipid extracted. Thus, single and tandem MS measurements were performed on a hybrid quadrupole-Orbitrap mass spectrometer for the characterization of fibroblast membrane lipids with the aim to apply this strategy for successive biomarker discover in PD patients.

Keywords

lipid extraction, HILIC, tandem MS, fibroblasts lipid biomarkers

Introduction

Lipids play multiple important roles being involved in energy pool, ensuring structural integrity of cellular membranes, regulation of membrane trafficking and signal transduction [1,2]. The study of the role of lipids has been complicated by their heterogeneity; they are so numerous in terms of fatty acyls and classes: glycerolipids, glycerophospholipids (GP), sphingolipids, sterol lipids, prenol lipids, saccharolipids and polyketides [3]. The number of theoretically possible structures is impressive also in view of the existence of double bond positional isomers, sn-positional isomers, different double bond stereochemistry and different chirality. Overall, mammalian cells may contain about 1000 to 2000 lipid species, not including oxidized or damaged lipids as well [4]. Lipidomics, a branch of metabolomics, aims at the full characterization of lipids in biological samples as fluids, tissues, cells with goals such as: (i) finding of specific biomarkers for risk and disease diagnosis on a large scale, (ii) elucidation of altered lipid pathways indicative of disease, environmental perturbations or response to diet, drugs and toxins as well as genetics [5]. The above mentioned structural diversity of lipids signifies a serious challenge in the field of analytical chemistry. Fortunately, recent advancements in mass spectrometry (MS) soft ionization methods, such as MALDI and ESI with related instrumentation have greatly facilitated lipidomics studies and their fall out in understanding complex lipid functions. ESI-MS, and especially its coupling with chromatographic separations, is the most widely used technique in lipidomics. The MS analysis of lipids in biological samples has provided abundant data for clinical clarifications of many diseases. For instance, it is nowadays clear that important neurological disorders involve dysregulation in lipid metabolism [6]. Parkinson's disease (PD) is a chronic, progressive neuropsychiatric disease which affects the dopaminergic nigrostriatal pathway [7,8]. PD patients begin to manifest primary symptoms when 50%-80% of the nigrostriatum has degenerated [9], resulting in late treatments and ineffective prognosis. Molecular studies on the genes responsible for familial PD including α-synuclein (PARK1), Parkin (PARK2), and PINK1 (PARK6) [10,11] showed a strong connection between PD and mitochondria, pointing out that mitochondrial dysfunctions and resulting oxidative cellular stress may be crucial to PD pathogenesis [12,13]. More recent accumulated evidences demonstrated that altered cerebral lipid homeostasis had a significant effect on neurodegenerative pathways in PD. Genetic deficit of α-synuclein in mice results in increased levels of neutral lipids like cholesterol, cholesteryl esters and triacylglycerols [14]; changes in phospholipids levels have been observed in old transgenic mice expressing human α-synuclein [15]. Other studies have suggested changes in cerebral cholesterol, oxysterols, and cholesterol hydroperoxides during PD progression [16] or substantial changes in sphingolipid and glycerophospholipid biosynthetic pathways in the visual cortex of PD patients [17].

Currently, direct evidence of these pathological processes can be obtained prevalently in postmortem brain tissues. Numerous studies have been carried out in peripheral cells as lymphomonocytes, platelets, red blood cells but results have been often contradictory and, in most cases, strongly biased by pharmacological treatments [18]. Fibroblasts share the same genetic complexity of neurons and can represent an easily available substrate to investigate PD mechanisms [19,20]. However, there are still some limitations about current methods and the development of novel methodology for better understanding of lipid molecular species is still a hot topic [21]. The goal of this study was, therefore, to develop a lipidomics study on primary dermal fibroblasts derived from healthy controls with the final aim to apply this strategy to Parkinson's disease patients to find early biomarkers associated with neurodegeneration in familial PD.

Materials and Methods

Chemicals

Water, acetonitrile, methanol, chloroform, formic acid, methyl-tert-butyl- ether (MTBE), and ammonium acetate were obtained from Sigma-Aldrich (Milan, Italy). Normal human dermal fibroblasts (NHDF) from adult persons were purchased from Carlo Erba (Milano, Italy). Standard lipids were purchased from Spectra 2000 SRL (Rome, Italy). All solvents used were LC-MS grade except for CHCl3 and MTBE (HPLC grade). Calibrating solution containing caffeine, methionine-arginine-phenylalanine-alanine peptide and Ultramark, a mixture of fluorinated phosphazines, for positive and negative calibrations were purchased from Thermo Scientific (Waltham, Massachusetts, United States).

Sample Preparation

Cell growth

Age-matched adult normal human dermal fibroblasts (NHDF) were used as healthy controls. The cells were washed in PBS (phosfate buffered saline) and then centrifuged at 500 × g for 4 min at 25°C. The pellet was resuspended in the culture medium for subsequent lipid extraction.

Lipid extraction

Because the efficiency of simultaneously extracting various categories of lipids differs among methods, four protocols described in the following, were tested on human fibroblasts.

  1. Bligh Dyer protocol: Following the Bligh &Dyer protocol [22], 3 mL of methanol/chloroform (2:1, v/v) were added to 50 µL of fibroblast (FB) sample homogenate diluted with 750 µL of water and the mixture was left one hour at room temperature. Then, 1 mL of chloroform was added, and the mixture was vortexed for 30 s. Finally, upon adding 1 mL of water the solution was shaken before being centrifuged for 10 min at 2000 rpm. The lower phase containing lipids was dried under nitrogen; the residue was dissolved in 50 µL of methanol and then analyzed by LC-MS.
  2. Methanol/chloroform/MTBE: Recently, Pellegrino et al. [23] proposed a one-phase extraction (OPE) method based on a mixture of methanol/chloroform/MTBE (MMC) in 1.33/1/1 (v/v/v) ratio for the extraction of lipid classes from human serum. Following this OPE protocol, 50 μL of FB sample homogenate were incubated with 1 mL of the solvents mixture, then vortexed, shaken for 20 minutes before being centrifuged for 5 min at 3000 rpm. The organic phase was collected, dried and resuspended in 50 µL of methanol and then analyzed by LC-MS.
  3. 2-methoxy-2-methylpropane (MBTE): Lipids were extracted according to 2-methoxy-2- methylpropane (also known as methyl-tert-butyl-ether, MTBE) protocol [24] by adding 200 µL of chloroform and 400 µL of methanol to 50 µL of FB sample and vortex mixing for 5 min. Afterwards, 200 µL of chloroform were added followed by vortex mixing for 30 s. Finally, 200 µL of water were added and the solution centrifuged for 5 min at 1000 rpm. The final biphasic system could separate into two layers and the lower phase was collected. The lipid extract was dried, and the residue was dissolved in 50 µL of methanol.
  4. Butan-1-ol (BuOH) extraction procedure: In this procedure proposed by Baker et al. [25], 50 µL of FB cell homogenate were diluted with 450 µL of water and mixed with 60 µL of buffer containing 200 mM citric acid and 270 mM disodium hydrogenphosphate (pH= 4). Extraction was performed with 1 mL of 1-butanol and 500 µL of water-saturated butan-1-ol. The recovered butanol phase was evaporated to dryness under reduced pressure. The residue was dissolved back in 50 µL of ethanol.

HILIC-ESI-MS instrumentation and operating conditions

HILIC-ESI-FTMS measurements were performed using an LC-MS apparatus consisting in an UHPLC system. Ultimate 3000 and a hybrid Q-Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA), equipped with a heated electrospray ionization (HESI) source and a higher collisional energy dissociation (HCD) cell for tandem MS analyses. Chromatographic separations were run at ambient temperature (22 ± 1°C) on a narrow-bore Ascentis Express HILIC column (150 × 2.1 mm ID, 2.7 μm particle size) equipped with an Ascentis Express HILIC (5 × 2.1 mm ID) security guard cartridge (Supelco, Bellefonte, PA, USA) using a flow rate of 0.3 mL min-1. A volume of 5 μL of FB lipid extract was injected into the column using a RS Autosampler (Thermo Scientific, Waltham, MA, USA). The adjusted binary elution program, based on water and 2.5 mmol l-1 (solvent A) and acetonitrile (solvent B), both containing 0.1% (v/v) of formic acid, was adopted: 0-5 min, linear gradient from 97 to 88% solvent B; 5-10 min, isocratic at 88% solvent B; 10-11 min, linear gradient from 88 to 81% solvent B; 11-20 min, linear gradient from 81 to 70% solvent B; 20-22 min, linear gradient from 70 to 50% solvent B; 22-28 isocratic at 50% solvent B; 28-30 min, return to the initial composition, followed by a 5 min equilibration time.

The column effluent was transferred into the qExactive spectrometer through the HESI source. The main ESI and ion optic parameters were the following: sheath gas flow rate, 35 (arbitrary units); auxiliary gas flow rate, 15 (arbitrary units); spray voltage, 3.5 kV (positive) and -2.5 kV in negative polarity; capillary temperature, 320°C; S-lens radio frequency level, 100 (arbitrary units). Negative and positive MS full-scan spectra were acquired in the m/z range 130-2000, after setting a mass resolving power of 140 000 (at m/z 200). During MS measurements, the Orbitrap fill time was set to 200 ms and the automatic gain control (AGC) level to 3 × 106. The Q Exactive spectrometer was daily calibrated and mass accuracies ranged between 0.10 and 0.17 ppm in positive polarity and between 0.40 and 0.45 ppm in negative polarity.

To recover more structural information on lipid classes, additional MS acquisitions were concurrently performed adopting the so-called MS/AIF/NL dd-MS2 workflow implemented in the qExactive instrumentation. In this sequence, a high-resolution MS full scan is first performed, followed by an all ion fragmentation (AIF) scan, i.e., a scan of product ions generated in the HCD cell from all the ions coming from the HESI source. Spectra obtained from these two events are rapidly compared by software, and when a user-defined m/z neutral loss (NL) is found (tolerance 10 ppm), the corresponding precursor ion is subjected to data dependent MS/MS acquisition. AIF measurements were performed at 35% normalized collision energy (NCE) at a resolving power of 70000 (at m/z 200), using an Orbitrap fill time of 200 ms and setting the automatic gain control level to 3 × 106. In some cases, the confirmation of lipid species individuated after elaborating MS/AIF/NL dd-MS2 data was obtained performing further LC-MS runs using targeted-MS2 acquisitions. In this modality, the m/z values of the selected precursor ions were introduced into an inclusion list, each with a tolerance of 10 ppm. MS/MS measurements were performed, in both positive and negative polarities, using a and a 1 m/z unit wide window, a resolving power of 70 000 (at m/z 200), a fill time of 100 ms and AGC of 2 × 105. The control of the LC-MS instrumentation and the first processing of data were performed by the Xcalibur software, version 3.0.63 (Thermo Scientific). The post analyses data processing was performed by using SigmaPlot 11.0 to graph final mass spectra while ChemDraw Pro 8.0.3 (CambridgeSoft Corporation, Cambridge, MA, USA) was employed to draw chemical structures.

Results and Discussion

Standard PL Analysis by LC-ESI-MS/MS

The application of high performance liquid chromatography-mass spectrometry (HPLC–MS) in phospholipids (PLs) analysis by using normal phase (NP) or reverse phase chromatography (RPC) has been widely reported, though the results were not always directly comparable due to different experimental conditions employed. Generally, the mobile phases used for NPC such as chloroform or hexane lack compatibility with electrospray ionization (ESI) technique and are difficult to handle because of their toxicity and volatility [26]. Contrary, RPC guarantees the use of well compatible solvents even if the separation of lipids based on their lipophilicity (alkyl chain length and/or degree of saturation), does not show class distinction leading to very complex chromatograms. Due to the limitations of these methods a relatively novel hydrophilic liquid chromatography (HILIC) has been proposed for the separation of amphiphilic compounds such as phospholipids [27]. HILIC is considered a variant of NPC that uses a hydrophilic stationary phase and organic aqueous solvents as mobile phase to avoid the disadvantages of the NP mode, such as the toxicity and volatility of solvents. The presence of more than 80% acetonitrile in HILIC mobile phase, provides more efficient mobile phase desolvation and compound ionization improving the ESI analysis; moreover, a separation based on the lipid’s head group polarity simplifies the chromatograms of very complex samples. Different applications of HILIC in lipids analysis have been reported in various food or biological matrices [27–33].

In this study the separation method was developed using a fused-core HILIC narrow- bore (150 × 2.1 mm ID) column. The fused core columns offers a thin porous shell of high-purity silica neighbouring a solid silica core. This particle design displays high column efficiency due to the narrow diffusion paths in the 0.5-micron thick porous shell and the small total particle size of 2.7 microns. The addition of 2.5 mM/L ammonium acetate containing 0.1% formic acid to the mobile phase contributes to improve the ionization of PLs in ESI Ms. In the current study, the retention time window of diverse lipid classes was firstly stated by appropriate lipid standards. PLs with head groups of choline (PC), ethanolamine (PE), serine (PS), inositol (PI) and sphingomyelin (SM) were selected. Figure 1 shows the total ion chromatogram (TIC) of the mixture of seven lipids namely PI (16:0/16:0), PE (16:0/18;1), PS (16:0/16:0), PC (12:0/12:0), PC (18:0/18:1), SM (42:2) and LPC (18:1/0:0) obtained in negative (A) and positive (B) ion mode.

Figure 1. Total ion chromatogram (TIC) of the mixture of seven lipids namely PI (16:0/16:0), PE (16:0/18;1), PS (16:0/16:0), PC (12:0/12:0), PC (18:0/18:1), SM (42:2) and LPC (18:1/0:0) obtained in negative (A) and positive (B) ion mode.

Applying the gradient elution program described in the experimental section, the separation of each lipid class was achieved in approximately in 20 min. The elution order, reflecting the increasing polarity, is: PI, PE, PS, PC (36:1), PC (24:0), SM and LPC. As expected, since LPC have only one acyl chain with the other acyl chain replaced by a hydroxyl group in sn-1 or sn-2 position, it elutes as the last species being the most polar. It can be noted that the separation of PS remains challenging probably due to the complexity of its retention mechanism in HILIC originating from a combined effect of hydrophilic and electrostatic (ionic) interactions caused by the amino acid on the polar head. PS elutes as a broad band starting around 13 minutes and continuing until 25 minutes. It is likely that at the beginning of the gradient the charged PS species interacts more with weakly acidic ionized silanols of the bare silica. This interaction decreases during gradient due to the competing formate ions originating from the increasing ammonium formate in aqueous mobile phase (at 13 minutes water reached around 20%) making possible a partitioning between the layer of water on the surface and the bulk organic enriched mobile phase [34]. From the TIC profile it is also clear a separation in the PC subclass with elution order depending on the type of fatty acids esterified at the glycerol backbone. The comparison among the two ionization modalities highlights that PCs, SM and LPCs were detected more easily in positive ion mode while PE, PI and PS were ionized preferentially in negative ion mode. However, in our case, PCs, SMs and LPCs were also well detected in negative ion mode as formate ([M+HCOO]), acetate ([M+CH3COO]), or demethylated ([M-CH3]) adducts. Indeed, a preliminary source-induced fragmentation (sid) was applied at 40 eV of collisional energy to enhance the generation of [M-CH3]− ions diagnostic of these PLs bearing a choline moiety in the polar head.

Comparison of lipid extraction protocols

The most broadly used protocols for lipid extraction are those of Folch et al. [35] and Bligh and Dyer [22] often in somewhat modified versions [36,37]. However, new alternative methods have been recently proposed with the main scope to reduce the use of toxic and carcinogenic halogenated solvents (e.g., chloroform). Granafei et al. [38] demonstrated that an extraction protocol based on MTBE (2- methoxy-2-methylpropane) provides similar recoveries to Bligh Dyer for all major lipid classes from fish brain tissue. The MTBE-based protocol has also been applied for PL study in plasma [24] and human occipital cortex [39]. However, MTBE could not be able to dissolve highly polar lipids and its low flash point requires additional attention for solvent handling [38]. Other two protocols based on butanol [25] or single phase MMC [23] were useful for the analysis of lipids in human plasma and serum showing a better extraction of phosphatidic acid (PA) or PG [25] or neutral lipids [23], respectively. We examined these four protocols on FB cells with the aim to understand which one can allow a more comprehensive extraction in terms of number of lipids using the previous optimized LC-ESI MS/MS. The FB cell homogenate sample was extracted in triplicate using each protocol (see experimental section) and Figure 2 summarizes TICs of FB lipid extract relevant to MTBE (A), BuOH (B), MMC (C) and BD (D) protocol. The elution order observed for PL classes reflects the separation attained using standard lipids being PI, PE, PS, PC and SM the main classes retrieved. A snapshot about the lipid composition of a given PL class can be obtained looking at the MS spectrum averaged over the entire HILIC band related to that class. Figure 3 reports the spectra relevant to PC (A) and SM (B) in positive ion mode, PI (C) and PE (D) in negative ion mode averaged under the chromatographic bands of BD extract. As can be seen, very similar results were obtained in the total number of extracted lipids comparing the four protocols. However, even if all the lipid species were retrieved under each spectrum significant differences were related to the presence of interfering compounds (overall for MMC protocol) not recognized as lipid species from the database search. The presence of co-eluting interfering peaks can represent a problem for a reliable quantification of specific compounds. A method to estimate the contribution of subspecies from different classes to the peak shape can be the calculation of tailing factor (TF). The TF values should normally fall between 1.0 and 1.5 for an optimized method [34]. We calculated TF for each protocol by using the eXtracted Ion Current (XIC) trace on the most abundant PC at 760.585 m/z (± 2.5 ppm) reported in Figure 4. The TF was obtained with the following formula: TF =?/2β where ? is the peak width at 5% peak height and β is the difference between the retention time at the beginning of the peak (at 5% peak height) and the retention time at the peak maximum. The value of TF was acceptable for MTBE (1.4) and BD (1.5) protocols while it was higher for BuOH (1.8) and MMC (2.1). However, MTBE protocol originates chromatograms with lower S/N ratio, so the BD protocol was selected for further extractions.

Figure 2. Total ion chromatograms (TICs) of fibroblast lipid extract relevant to MTBE (A), BuOH (B), MMC (C) and BD (D) protocol.

Figure 3. FTMS spectra relevant to PC (A) and SM (B) in positive ion mode, PI (C) and PE (D) in negative ion mode averaged under the chromatographic bands of Bligh Dyer extract.

Figure 4. Extracted Ion Current (XIC) traces on the most abundant PC at 760.585 m/z (± 2.5 ppm) obtained by HILIC-ESI(−)-FTMS of the MTBE (A), BuOH (B), MMC (C) and BD (D) lipid extracts.

Characterization of lipid species in FB extract

The characterization of the lipid species detected under each band was carried out. Besides the accurate m/z value we utilized the already mentioned AIF signal acquisition approach, provided by FTMS instrumentation, that helped in the recognition of the main PL classes thanks to the generation of specific class- related product ions without the necessity to isolate and fragment certain precursor ions. By exploiting the high collisional energies provided by the HCD cell, the informative fragment ions generated from the head group were used to identify the lipid class of interest.

For example, the XIC obtained for the BD lipid extract using AIF acquisition is reported in Figure 5 for negative (A) and positive polarity (B). The first ion extracted at m/z 196.0375 (±0.0015) corresponds to the molecular formula [C5H11NO5P] (structure reported in the inset of Figure 5A) related to a product ion obtained from PLs bearing a phosphoethanolamine head. The ion extracted at m/z 184.0733 (±0.0015) corresponds to the exclusive fragment of phosphocholine (structure reported in the inset of Figure 5B) belonging both to PC and SM. The extremely narrow window chosen led to the elimination of any interference due to contaminant ions. By following this approach 266 lipid species with various regiochemistry were identified from main lipid classes and molecular formulas are presented in Table 1. From the analysis of the spectra collected we noticed that very few lysoPLs (LPL) were retrieved. Usually these species can be clinically important potential biomarkers of pathology since they are mostly produced by the enzymatic reaction of phospholipases, involved in signalling mechanisms and in the pathway of disease development [40,41]. Therefore, the determination of LPLs can be of interest to establish the relationship of diseases and the changes in composition and concentration also in fibroblasts. However, attention should be paid on this point since LPL can be also markers of sample storage and handling procedures as recently reported for fish brain [38]. To this aim we decided to re-extract and re-analyzed lipids from FB cells after storing samples for three months at -20ºC. Figure 6 reports the XIC for LPC 18:1 at m/z 522. 325 (±0,025 m/z) for fresh (A) and old (B) sample and LPE 18:0 at m/z 502. 295 (±0,025 m/z) for fresh (C) and old (D) sample. The absolute signal intensity but also the ratio to the main PC and PE (data not shown) demonstrate that the level of LPLs was increased of almost two orders of magnitude on old sample thus confirming that the sample age greatly influences LPL/PL ratio. The XIC of the selected LPC shows two contributions labelled as a and b in Figure 6. The chosen gradient allowed to separate LPC regioisomers which differs by the position of the acyl chain attached to the glycerol carbon. The relevant MS/MS on the protonated compound [M+H]+ were subjected to HCD-MS/MS examination and results are reported in Figure 7A and 7B. The regiochemistry of LPC species can be easily assessed exploiting the presence of the ion at m/z 104.11 assigned to the choline ion that is reported to be more intense for sn1 position [42]. In this way it was also possible to assign the compound as LPC (0:0/18:1) for peak a and LPC (18:1/0:0) for peak b.

Table 1. Overview of phospholipids identified in fibroblast cells by HILIC-ESI-FTMS in negative ion mode.

 

Experimental m/z

Formula

Adduct

Attribution

PC_1

660.4609

C35H67NO8P

[M-CH3]

12:0_16:1

PC_2

662.513

C35H67NO8P

[M-CH3]

12:0_16:0; 14:0/14:0

PC_3

674.4766

C36H69NO8P

[M-CH3]

15:0_14:1; 15:1_14:0;

PC_4

690.5078

C37H73NO8P

[M-CH3]

14:0/16:0; 15:0/15:0

PC_5

700.4922

C38H71NO8P

[M-CH3]

17:1_14:0; 16:0_15:1;

PC_6

700.5079

C38H73NO8P

[M-CH3]

15:1/16:0

PC_7

702.5079

C38H73NO8P

[M-CH3]

16:1_15:0

PC_8

704.5235

C38H75NO8P

[M-CH3]

15:0/16:0; 14:0-17:0

PC_9

714.5079

C39H73NO8P

[M-CH3]

16:1/16:1; 18:2_14:0

     

16:2_16:0; 18:1_14:1

PC_10

716.5235

C39H75NO8P

[M-CH3]

16:0/16:1; 18:1_14:0

PC_11

718.5392

C39H77NO8P

[M-CH3]

16:0/16:0; 18:0_14:0

PC_12

728.5235

C40H75NO8P

[M-CH3]

17:1/16:1; 15:1-18:1; 15:0-18:2;

     

17:0-16:2; 17:1-16:1; 14:0-19:2

PC_13

730.5392

C40H77NO8P

[M-CH3]

16:0_17:1; 16:1_17:0; 18:1_15:0

PC_14

732.5548

C40H79NO8P

[M-CH3]

16:0/17:0

PC_15

738.5079

C41H73NO8P

[M-CH3]

20:3-14:1; 18:2-16:2; 16:1-18:3; 14:0-20:4

PC_16

742.5392

C41H77NO8P

[M-CH3]

16:1/18:1;16:0_18:2

PC_17

744.556

C41H79NO8P

[M-CH3]

16:0/18:1

PC_18

746.5705

C41H81NO8P

[M-CH3]

16:0/18:0

PC_19

748.5142

C39H75NO10P

[M+HCOO]

14:0/16:1

PC_20

750.5303

C39H77NO10P

[M+HCOO]

14:0/16:0

PC_21

752.536

C41H73NO8P

[M-CH3]

14:0/20:4

PC_22

756.5549

C42H79NO8P

[M+HCOO]

18:1/17:1; 16:0-19:2; 18:1-17:1; 16:1-19:1

PC_23

758.5705

C42H81NO8P

[M-CH3]

17:0/18:1; 17:1_18:0; 16:0_19:1

PC_24

764.5235

C43H75NO8P

[M-CH3]

18:1_20:4; 16:0_22:5

PC_25

770.5705

C43H81NO8P

[M-CH3]

18:1/18:1

PC_26

772.5861

C43H83NO8P

[M-CH3]

18:0_18:1

PC_27

774.5297

C41H77NO10P

[M+HCOO]

16:1/16:1

PC_28

776.5459

C41H79NO10P

[M+HCOO]

16:0/16:1; 18:1_14:1; 15:0/17:1

PC_29

778.5612

C41H81NO10P

[M+HCOO]

15:0/17:0; 16:0/16:0

PC_30

782.5705

C44H81NO8P

[M-CH3]

17:0_20:3

PC_31

784.5861

C44H83NO8P

[M-CH3]

19:1_18:1; 18:2_19:0; 18:0_19:2

PC_32

788.5811

C43H83NO9P

[M-CH3]

16:1/18:1; 18:1/16:1

PC_33

792.5548

C45H79NO8P

[M-CH3]

16:0-22:5; 18:2-20:3; 18:1-20:4; 16:1-22:4

PC_34

794.5705

C45H81NO8P

[M-CH3]

18:1-20:3; 18:0-20:4; 16:0-22:4

PC_35

796.5861

C45H83NO8P

[M-CH3]

18:0-20:3; 18:1-20:2; 18:2-20:1

     

16:0-22:3; 16:1-22:2

PC_36

798.6018

C45H85NO8P

[M-CH3]

18:1/20:1; 18:0/20:2; 16:2_22:0

PC_37

804.576

C43H83NO10P

[M+HCOO]

16:0/18:1

PC_38

810.6018

C46H85NO8P

[M-CH3]

18:0_21:3

PC_39

816.5548

C47H79NO8P

[M-CH3]

18:1-22:6; 18:2-22:5; 20:3-20:4

PC_40

818.5705

C47H81NO8P

[M-CH3]

18:1-22:5; 18:0-22:6; 18:2-22:4

     

20:3-20:3; 20:2-20:4

PC_41

820.5861

C47H83NO8P

[M-CH3]

18:0-22:5; 18:1-22:4; 20:2-20:3; 20:4-20:1

PC_42

826.5621

C45H81NO10P

[M+HCOO]

16:0/20:4

PC_43

830.5924

C45H85NO10P

[M+HCOO]

18:1/18:1; 18:0_18:2; 16:0_20:2

PC_44

832.6073

C45H87NO10P

[M+HCOO]

18:0/18:1; 19:0_16:1

PC_45

846.6229

C46H89NO10P

[M+HCOO]

18:1/19:0; 19:1_18:0; 20:0_17:1

PC_46

854.5922

C47H85NO10P

[M+HCOO]

18:0/20:4; 18:1/20:3; 16:0_22:4

       

PE_1

688.4922

C37H71NO8P

[M-H]

16:0/16:1; 18:1/14:0

PE_2

716.5235

C39H75NO8P

[M-H]

16:0/18:1; 18:1/16:0

PE_3

736.4922

C41H71NO8P

[M-H]

16:1/20:4; 16:0_20:5; 14:0_22:5

PE_4

738.5079

C41H73NO8P

[M-H]

16:0_20:4; 16:1_20:3

PE_5

740.5235

C41H75NO8P

[M-H]

18:1/18:2;

PE_6

742.5417

C41H77NO8P

[M-H]

18:1/18:1; 18:0/18:3

PE_7

744.5548

C41H79NO8P

[M-H]

18:0/18:1

PE_8

752.5235

C42H75NO8P

[M-H]

17:1/20:3; 17:0_20:4

PE_9

762.5079

C43H73NO8P

[M-H]

18:1/20:5; 16:0_22:6; 16:1_22:5

PE_10

764.5245

C43H75NO8P

[M-H]

18:1/20:4

PE_11

766.539

C43H77NO8P

[M-H]

18:0/20:4; 18:1_20:3

PE_12

768.5548

C43H79NO8P

[M-H]

18:0/20:3; 18:1/20:2

PE_13

770.5705

C43H81NO8P

[M-H]

18:0/22:0

PE_14

776.5235

C44H75NO8P

[M-H]

17:1/22:5

PE_15

790.5395

C45H77NO8P

[M-H]

18:0/22:6

PE_16

792.5548

C45H79NO8P

[M-H]

18:0/22:5; 18:1_22:4

       

LPC_1

452.2782

C21H43NO7P

[M-CH3]

14:0/0:0

LPC_2

466.2938

C22H45NO7P

[M-CH3]

15:0/0:0

LPC_3

478.2938

C23H45NO7P

[M-CH3]

0:0/16:1

LPC_4

480.3095

C23H47NO7P

[M-CH3]

0:0/16:0

LPC_5

492.3095

C24H47NO7P

[M-CH3]

17:1/0:0

LPC_6

494.3251

C24H49NO7P

[M-CH3]

17:0/0:0

LPC_7

504.3095

C25H47NO7P

[M-CH3]

0:0/18:2

LPC_8

506.3251

C25H49NO7P

[M-CH3]

0:0/18:1

LPC_9

508.3408

C25H51NO7P

[M-CH3]

0:0/18:0

LPC_10

526.2938

C27H45NO7P

[M-CH3]

0:0/20:5

LPC_11

534.3564

C27H53NO7P

[M-CH3]

0:0/20:1

LPC_12

536.3721

C27H55NO7P

[M-CH3]

0:0/20:0

LPC_13

618.3776

C33H65NO7P

[M-CH3]

0:0/26:1

LPC_14

620.466

C33H67NO7P

[M-CH3]

0:0/26:0

       

LPE_1

452.2782

C21H43NO7P

[M-H]

16:0

LPE_2

480.3095

C23H47NO7P

[M-H]

18:0

LPE_ 3

498.264

C25H41NO7P

[M-H]

20:5

LPE_4

506.3262

C25H49NO7P

[M-H]

20:1

LPE_5

526.2939

C27H45NO7P

[M-H]

22:5

LPE_6

528.3095

C27H47NO7P

[M-H]

22:4

LPE_7

536.3721

C27H55NO7P

[M-H]

22:0

LPE_8

562.3878

C29H57NO7P

[M-H]

24:1

       

PI_1

807.5092

C41H76O13P

[M-H]

16:0/16:1; 18:1_14:0; 18:0_14:1

PI_2

819.5206

C42H76O13P

[M-H]

18:1/15:1; 16:1_17:1

PI_3

831.5029

C43H76O13P

[M-H]

18:1/16:2; 16:1_18:2; 16:0_18:3

PI_4

833.5185

C43H78O13P

[M-H]

18:1/16:1; 16:0_18:2; 18:0_16:2

PI_5

835.5342

C43H80O13P

[M-H]

18:1/16:0; 16:1_18:0

PI_6

847.5342

C44H80O13P

[M-H]

18:1/17:1; 16:1_19:1; 18:0_17:2; 18:2_17:0;

     

16:0_19:2

PI_7

849.5498

C44H82O13P

[M-H]

18:1/17:0;1 7:1_18:0; 16:0_19:1; 16:1/19:0

PI_8

857.5183

C45H78O13P

[M-H]

16:0/20:4; 20:3-16:1; 18:1-18:3; 18:2-18:2

PI_9

859.5342

C45H80O13P

[M-H]

18:1/18:2; 18:0_18:3; 16:0-20:3; 20:2-16:1

PI_10

861.5498

C45H82O13P

[M-H]

18:1/18:1; 18:0_18:2; 16:0_20:2

PI_11

863.5655

C45H84O13P

[M-H]

18:1/18:0

PI_12

871.5342

C46H80O13P

[M-H]

17:0/20:4; 20:3_17:1

     

19:3_18:1; 18:0_19:4; 21:3_16:1

PI_13

873.5498

C46H82O13P

[M-H]

19:3/18:0; 18:1_19:2

     

17:0_20:3; 20:2/17:1; 16:0/21:3

PI_14

881.5185

C47H78O13P

[M-H]

18:1/20:5; 20:4_18:2; 16:0/22:6; 22:5/16:1

PI_15

883.3342

C47H80O13P

[M-H]

20:4/18:1; 20:3_18:2; 18:0_20:5; 16:0_22:5

PI_16

885.5498

C47H82O13P

[M-H]

18:0/20:4; 20:3_18:1; 16:0_22:4

PI_17

887.5655

C47H84O13P

[M-H]

18:0/20:3; 18:1-20:2

PI_18

897.5498

C48H82O13P

[M-H]

18:1/21:4; 21:3_18:2; 18:0_21:5; 17:1_22:4;

     

19:1_20:4; 20:3_19:2; 19:0_20:5

PI_19

899.5655

C48H84O13P

[M-H]

21:3/18:1; 18:0_21:4; 19:1_20:3; 20:4_19:0;

     

19:2_20:2; 17:1_22:3

PI_20

907.5342

C49H80O13P

[M-H]

18:1/22:6; 18:2-22:5; 20:4-20:3

PI_21

909.5502

C49H82O13P

[M-H]

18:1/22:5; 18:0_22:6; 18:2_22:4

PI_22

911.5648

C49H84O13P

[M-H]

18:0/22:5; 20:1_20:4; 18:1_22:4

PI_23

913.5811

C49H86O13P

[M-H]

18:0/22:4; 18:1_22:3; 20:1_20:3; 20:2_20:2;

SM_1

685.5302

C39H79N2O6P

[M-CH3]

Cer d18:1/16:0; Cer d18:0/16:1

SM_2

687.5454

C39H81N2O6P

[M-CH3]

Cer d18:1/16:0

SM_3

689.5504

C39H81N2O6P

[M-CH3]

Cer d18:0/16:1;Cer d18:1/16:0

SM_4

701.5604

C41H83N2O6P

[M-CH3]

Cer d18:1/17:0;Cer d19:1/16:0

 

747.5672

C41H83N2O6P

[M+HCOO]

Cer d18:1/17:0; Cer d19:1/16:0

SM_5

713.5609

C41H83N2O6P

[M-CH3]

Cer d18:2/18:0; Cer d18:1/18:1

SM_6

715.577

C41H85N2O6P

[M-CH3]

Cer d18:0/18:1

SM_7

729.593

C42H85N2O6P

[M-CH3]

Cer d18:1/19:0; Cer d19:1/18:0;

     

Cer d19:0/18:1; Cer d17:1/20:0

SM_8

741.5935

C43H85N2O6P

[M-CH3]

Cer d18:2/20:0; Cer d18:1/20:1

SM_9

769.6241

C45H89N2O6P

[M-CH3]

Cer d18:1/22:1; Cer d18:2/22:0

SM_10

771.6398

C45H91N2O6P

[M-CH3]

Cer d18:1/22:0; Cer d22:0/18:1

SM_11

783.6405

C46H91N2O6P

[M-CH3]

Cer d18:1/23:1; Cer d17:1/24:1

SM_12

797.6556

C47H93N2O6P

[M-CH3]

Cer d18:1/24:1; Cer d18:2/24:0

 

857.6766

C47H93N2O6P

[M+CH3COO]

Cer d18:1/24:1; Cer d18:2/24:0

Figure 5. Extracted Ion Current (XIC) traces obtained for the BD lipid extract using AIF acquisition is reported in Figure 5 for negative (A) and positive polarity (B). The first ion extracted at m/z 196.0375 (±0.0015) is diagnostic of PE class while the ion at m/z 184.0733 (±0.0015) is diagnostic for PC, SM, and LPC. Some possible structures of these ions are given in the insets.

Figure 6. XIC chromatograms for LPC 18:1 centered at m/z 522. 325 (±0.025 m/z) for fresh (A) and stored (B) sample and LPE 18:0 at m/z 502. 295 (±0,025 m/z) for fresh (C) and stored (D) sample.

Figure 7. Tandem MS spectra of the proton adducts ([M+H]+) of regioisomeric LPC 0:0/18:1 (A) and 18:1/0:0 (B) at m/z 522.325 and LPE 18:0 (C) with m/z 502.295.

For LPE only one peak was detected in the chromatogram and it was assigned as LPE 18:0 (Figure 7C) but without assessing the regiochemistry. The LPLs identified in old samples were assigned as listed in Table 1. This last point highlights that the strategy developed could be easily applied to fibroblast cells for Parkinson patients provided that the collection, treatment and analysis of the cells are carried out simultaneously for the two samples typology.

Conclusions

The approach here proposed, shoots for overcoming the limits of conventional research approach in the field of neurodegenerative diseases where analyses are usually performed on postmortem brain tissues. To this aim, the peripheral fibroblasts have been studied as target samples since they can represent an accessible source of cells that share the same genetic information of neurons. After selecting the Bligh Dyer protocol as the most informative, HILIC- ESI-FTMS analyses, complemented by AIF MS2 experiments, performed at higher collisional dissociation energy regimes, have enabled the characterization of phospholipids in fibroblast cells. Here, for the first time, 266 main phospholipids were identified namely 104 PC, 28 PE, 14 LPC, 8 LPE, 84 PI and 28 SM. In the future minor lipids will be characterized after using specific sample pre-treatments. The overview of all lipids in whole cells is the starting point for a targeted lipidomics approach (characterization and quantification) that will be performed also in fibroblasts from skin biopsies of patients with Parkinson's disease.

Acknowledgments

This work was supported by Fondazione Puglia in the framework of the project entitled “Sviluppo ed uso di tecniche avanzate di spettrometria di massa per la caratterizzazione del profilo lipidomico cellulare e mitocondriale in fibroblasti controllo e di pazienti affetti da morbo di Parkinson”.

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Editorial Information

Editor-in-Chief

Article Type

Research Article

Publication history

Received: 9 July, 2018
Accepted date: 26 July, 2018
Published date: 30 July, 2018

Copyright

©2018 Calvano CD. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Calvano CD (2018) Development and use of advanced mass spectrometry techniques for the characterization of cellular and mitochondrial lipidomic profiling in control fibroblasts and Parkinson's disease patients. Trends Pharm Biomed Anal 1: DOI: 10.15761/TPBA.1000102

Corresponding author

C D Calvano

Department of Chemistry, University of Bari Aldo Moro, via Orabona 4, 70126 Bari, Italy


A M Sardanelli

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari "Aldo Moro", P.zza G. Cesare11, 70124 Bari, Italy

Table 1. Overview of phospholipids identified in fibroblast cells by HILIC-ESI-FTMS in negative ion mode.

 

Experimental m/z

Formula

Adduct

Attribution

PC_1

660.4609

C35H67NO8P

[M-CH3]

12:0_16:1

PC_2

662.513

C35H67NO8P

[M-CH3]

12:0_16:0; 14:0/14:0

PC_3

674.4766

C36H69NO8P

[M-CH3]

15:0_14:1; 15:1_14:0;

PC_4

690.5078

C37H73NO8P

[M-CH3]

14:0/16:0; 15:0/15:0

PC_5

700.4922

C38H71NO8P

[M-CH3]

17:1_14:0; 16:0_15:1;

PC_6

700.5079

C38H73NO8P

[M-CH3]

15:1/16:0

PC_7

702.5079

C38H73NO8P

[M-CH3]

16:1_15:0

PC_8

704.5235

C38H75NO8P

[M-CH3]

15:0/16:0; 14:0-17:0

PC_9

714.5079

C39H73NO8P

[M-CH3]

16:1/16:1; 18:2_14:0

     

16:2_16:0; 18:1_14:1

PC_10

716.5235

C39H75NO8P

[M-CH3]

16:0/16:1; 18:1_14:0

PC_11

718.5392

C39H77NO8P

[M-CH3]

16:0/16:0; 18:0_14:0

PC_12

728.5235

C40H75NO8P

[M-CH3]

17:1/16:1; 15:1-18:1; 15:0-18:2;

     

17:0-16:2; 17:1-16:1; 14:0-19:2

PC_13

730.5392

C40H77NO8P

[M-CH3]

16:0_17:1; 16:1_17:0; 18:1_15:0

PC_14

732.5548

C40H79NO8P

[M-CH3]

16:0/17:0

PC_15

738.5079

C41H73NO8P

[M-CH3]

20:3-14:1; 18:2-16:2; 16:1-18:3; 14:0-20:4

PC_16

742.5392

C41H77NO8P

[M-CH3]

16:1/18:1;16:0_18:2

PC_17

744.556

C41H79NO8P

[M-CH3]

16:0/18:1

PC_18

746.5705

C41H81NO8P

[M-CH3]

16:0/18:0

PC_19

748.5142

C39H75NO10P

[M+HCOO]

14:0/16:1

PC_20

750.5303

C39H77NO10P

[M+HCOO]

14:0/16:0

PC_21

752.536

C41H73NO8P

[M-CH3]

14:0/20:4

PC_22

756.5549

C42H79NO8P

[M+HCOO]

18:1/17:1; 16:0-19:2; 18:1-17:1; 16:1-19:1

PC_23

758.5705

C42H81NO8P

[M-CH3]

17:0/18:1; 17:1_18:0; 16:0_19:1

PC_24

764.5235

C43H75NO8P

[M-CH3]

18:1_20:4; 16:0_22:5

PC_25

770.5705

C43H81NO8P

[M-CH3]

18:1/18:1

PC_26

772.5861

C43H83NO8P

[M-CH3]

18:0_18:1

PC_27

774.5297

C41H77NO10P

[M+HCOO]

16:1/16:1

PC_28

776.5459

C41H79NO10P

[M+HCOO]

16:0/16:1; 18:1_14:1; 15:0/17:1

PC_29

778.5612

C41H81NO10P

[M+HCOO]

15:0/17:0; 16:0/16:0

PC_30

782.5705

C44H81NO8P

[M-CH3]

17:0_20:3

PC_31

784.5861

C44H83NO8P

[M-CH3]

19:1_18:1; 18:2_19:0; 18:0_19:2

PC_32

788.5811

C43H83NO9P

[M-CH3]

16:1/18:1; 18:1/16:1

PC_33

792.5548

C45H79NO8P

[M-CH3]

16:0-22:5; 18:2-20:3; 18:1-20:4; 16:1-22:4

PC_34

794.5705

C45H81NO8P

[M-CH3]

18:1-20:3; 18:0-20:4; 16:0-22:4

PC_35

796.5861

C45H83NO8P

[M-CH3]

18:0-20:3; 18:1-20:2; 18:2-20:1

     

16:0-22:3; 16:1-22:2

PC_36

798.6018

C45H85NO8P

[M-CH3]

18:1/20:1; 18:0/20:2; 16:2_22:0

PC_37

804.576

C43H83NO10P

[M+HCOO]

16:0/18:1

PC_38

810.6018

C46H85NO8P

[M-CH3]

18:0_21:3

PC_39

816.5548

C47H79NO8P

[M-CH3]

18:1-22:6; 18:2-22:5; 20:3-20:4

PC_40

818.5705

C47H81NO8P

[M-CH3]

18:1-22:5; 18:0-22:6; 18:2-22:4

     

20:3-20:3; 20:2-20:4

PC_41

820.5861

C47H83NO8P

[M-CH3]

18:0-22:5; 18:1-22:4; 20:2-20:3; 20:4-20:1

PC_42

826.5621

C45H81NO10P

[M+HCOO]

16:0/20:4

PC_43

830.5924

C45H85NO10P

[M+HCOO]

18:1/18:1; 18:0_18:2; 16:0_20:2

PC_44

832.6073

C45H87NO10P

[M+HCOO]

18:0/18:1; 19:0_16:1

PC_45

846.6229

C46H89NO10P

[M+HCOO]

18:1/19:0; 19:1_18:0; 20:0_17:1

PC_46

854.5922

C47H85NO10P

[M+HCOO]

18:0/20:4; 18:1/20:3; 16:0_22:4

       

PE_1

688.4922

C37H71NO8P

[M-H]

16:0/16:1; 18:1/14:0

PE_2

716.5235

C39H75NO8P

[M-H]

16:0/18:1; 18:1/16:0

PE_3

736.4922

C41H71NO8P

[M-H]

16:1/20:4; 16:0_20:5; 14:0_22:5

PE_4

738.5079

C41H73NO8P

[M-H]

16:0_20:4; 16:1_20:3

PE_5

740.5235

C41H75NO8P

[M-H]

18:1/18:2;

PE_6

742.5417

C41H77NO8P

[M-H]

18:1/18:1; 18:0/18:3

PE_7

744.5548

C41H79NO8P

[M-H]

18:0/18:1

PE_8

752.5235

C42H75NO8P

[M-H]

17:1/20:3; 17:0_20:4

PE_9

762.5079

C43H73NO8P

[M-H]

18:1/20:5; 16:0_22:6; 16:1_22:5

PE_10

764.5245

C43H75NO8P

[M-H]

18:1/20:4

PE_11

766.539

C43H77NO8P

[M-H]

18:0/20:4; 18:1_20:3

PE_12

768.5548

C43H79NO8P

[M-H]

18:0/20:3; 18:1/20:2

PE_13

770.5705

C43H81NO8P

[M-H]

18:0/22:0

PE_14

776.5235

C44H75NO8P

[M-H]

17:1/22:5

PE_15

790.5395

C45H77NO8P

[M-H]

18:0/22:6

PE_16

792.5548

C45H79NO8P

[M-H]

18:0/22:5; 18:1_22:4

       

LPC_1

452.2782

C21H43NO7P

[M-CH3]

14:0/0:0

LPC_2

466.2938

C22H45NO7P

[M-CH3]

15:0/0:0

LPC_3

478.2938

C23H45NO7P

[M-CH3]

0:0/16:1

LPC_4

480.3095

C23H47NO7P

[M-CH3]

0:0/16:0

LPC_5

492.3095

C24H47NO7P

[M-CH3]

17:1/0:0

LPC_6

494.3251

C24H49NO7P

[M-CH3]

17:0/0:0

LPC_7

504.3095

C25H47NO7P

[M-CH3]

0:0/18:2

LPC_8

506.3251

C25H49NO7P

[M-CH3]

0:0/18:1

LPC_9

508.3408

C25H51NO7P

[M-CH3]

0:0/18:0

LPC_10

526.2938

C27H45NO7P

[M-CH3]

0:0/20:5

LPC_11

534.3564

C27H53NO7P

[M-CH3]

0:0/20:1

LPC_12

536.3721

C27H55NO7P

[M-CH3]

0:0/20:0

LPC_13

618.3776

C33H65NO7P

[M-CH3]

0:0/26:1

LPC_14

620.466

C33H67NO7P

[M-CH3]

0:0/26:0

       

LPE_1

452.2782

C21H43NO7P

[M-H]

16:0

LPE_2

480.3095

C23H47NO7P

[M-H]

18:0

LPE_ 3

498.264

C25H41NO7P

[M-H]

20:5

LPE_4

506.3262

C25H49NO7P

[M-H]

20:1

LPE_5

526.2939

C27H45NO7P

[M-H]

22:5

LPE_6

528.3095

C27H47NO7P

[M-H]

22:4

LPE_7

536.3721

C27H55NO7P

[M-H]

22:0

LPE_8

562.3878

C29H57NO7P

[M-H]

24:1

       

PI_1

807.5092

C41H76O13P

[M-H]

16:0/16:1; 18:1_14:0; 18:0_14:1

PI_2

819.5206

C42H76O13P

[M-H]

18:1/15:1; 16:1_17:1

PI_3

831.5029

C43H76O13P

[M-H]

18:1/16:2; 16:1_18:2; 16:0_18:3

PI_4

833.5185

C43H78O13P

[M-H]

18:1/16:1; 16:0_18:2; 18:0_16:2

PI_5

835.5342

C43H80O13P

[M-H]

18:1/16:0; 16:1_18:0

PI_6

847.5342

C44H80O13P

[M-H]

18:1/17:1; 16:1_19:1; 18:0_17:2; 18:2_17:0;

     

16:0_19:2

PI_7

849.5498

C44H82O13P

[M-H]

18:1/17:0;1 7:1_18:0; 16:0_19:1; 16:1/19:0

PI_8

857.5183

C45H78O13P

[M-H]

16:0/20:4; 20:3-16:1; 18:1-18:3; 18:2-18:2

PI_9

859.5342

C45H80O13P

[M-H]

18:1/18:2; 18:0_18:3; 16:0-20:3; 20:2-16:1

PI_10

861.5498

C45H82O13P

[M-H]

18:1/18:1; 18:0_18:2; 16:0_20:2

PI_11

863.5655

C45H84O13P

[M-H]

18:1/18:0

PI_12

871.5342

C46H80O13P

[M-H]

17:0/20:4; 20:3_17:1

     

19:3_18:1; 18:0_19:4; 21:3_16:1

PI_13

873.5498

C46H82O13P

[M-H]

19:3/18:0; 18:1_19:2

     

17:0_20:3; 20:2/17:1; 16:0/21:3

PI_14

881.5185

C47H78O13P

[M-H]

18:1/20:5; 20:4_18:2; 16:0/22:6; 22:5/16:1

PI_15

883.3342

C47H80O13P

[M-H]

20:4/18:1; 20:3_18:2; 18:0_20:5; 16:0_22:5

PI_16

885.5498

C47H82O13P

[M-H]

18:0/20:4; 20:3_18:1; 16:0_22:4

PI_17

887.5655

C47H84O13P

[M-H]

18:0/20:3; 18:1-20:2

PI_18

897.5498

C48H82O13P

[M-H]

18:1/21:4; 21:3_18:2; 18:0_21:5; 17:1_22:4;

     

19:1_20:4; 20:3_19:2; 19:0_20:5

PI_19

899.5655

C48H84O13P

[M-H]

21:3/18:1; 18:0_21:4; 19:1_20:3; 20:4_19:0;

     

19:2_20:2; 17:1_22:3

PI_20

907.5342

C49H80O13P

[M-H]

18:1/22:6; 18:2-22:5; 20:4-20:3

PI_21

909.5502

C49H82O13P

[M-H]

18:1/22:5; 18:0_22:6; 18:2_22:4

PI_22

911.5648

C49H84O13P

[M-H]

18:0/22:5; 20:1_20:4; 18:1_22:4

PI_23

913.5811

C49H86O13P

[M-H]

18:0/22:4; 18:1_22:3; 20:1_20:3; 20:2_20:2;

SM_1

685.5302

C39H79N2O6P

[M-CH3]

Cer d18:1/16:0; Cer d18:0/16:1

SM_2

687.5454

C39H81N2O6P

[M-CH3]

Cer d18:1/16:0

SM_3

689.5504

C39H81N2O6P

[M-CH3]

Cer d18:0/16:1;Cer d18:1/16:0

SM_4

701.5604

C41H83N2O6P

[M-CH3]

Cer d18:1/17:0;Cer d19:1/16:0

 

747.5672

C41H83N2O6P

[M+HCOO]

Cer d18:1/17:0; Cer d19:1/16:0

SM_5

713.5609

C41H83N2O6P

[M-CH3]

Cer d18:2/18:0; Cer d18:1/18:1

SM_6

715.577

C41H85N2O6P

[M-CH3]

Cer d18:0/18:1

SM_7

729.593

C42H85N2O6P

[M-CH3]

Cer d18:1/19:0; Cer d19:1/18:0;

     

Cer d19:0/18:1; Cer d17:1/20:0

SM_8

741.5935

C43H85N2O6P

[M-CH3]

Cer d18:2/20:0; Cer d18:1/20:1

SM_9

769.6241

C45H89N2O6P

[M-CH3]

Cer d18:1/22:1; Cer d18:2/22:0

SM_10

771.6398

C45H91N2O6P

[M-CH3]

Cer d18:1/22:0; Cer d22:0/18:1

SM_11

783.6405

C46H91N2O6P

[M-CH3]

Cer d18:1/23:1; Cer d17:1/24:1

SM_12

797.6556

C47H93N2O6P

[M-CH3]

Cer d18:1/24:1; Cer d18:2/24:0

 

857.6766

C47H93N2O6P

[M+CH3COO]

Cer d18:1/24:1; Cer d18:2/24:0

Figure 1. Total ion chromatogram (TIC) of the mixture of seven lipids namely PI (16:0/16:0), PE (16:0/18;1), PS (16:0/16:0), PC (12:0/12:0), PC (18:0/18:1), SM (42:2) and LPC (18:1/0:0) obtained in negative (A) and positive (B) ion mode.

Figure 2. Total ion chromatograms (TICs) of fibroblast lipid extract relevant to MTBE (A), BuOH (B), MMC (C) and BD (D) protocol.

Figure 3. FTMS spectra relevant to PC (A) and SM (B) in positive ion mode, PI (C) and PE (D) in negative ion mode averaged under the chromatographic bands of Bligh Dyer extract.

Figure 4. Extracted Ion Current (XIC) traces on the most abundant PC at 760.585 m/z (± 2.5 ppm) obtained by HILIC-ESI(−)-FTMS of the MTBE (A), BuOH (B), MMC (C) and BD (D) lipid extracts.

Figure 5. Extracted Ion Current (XIC) traces obtained for the BD lipid extract using AIF acquisition is reported in Figure 5 for negative (A) and positive polarity (B). The first ion extracted at m/z 196.0375 (±0.0015) is diagnostic of PE class while the ion at m/z 184.0733 (±0.0015) is diagnostic for PC, SM, and LPC. Some possible structures of these ions are given in the insets.

Figure 6. XIC chromatograms for LPC 18:1 centered at m/z 522. 325 (±0.025 m/z) for fresh (A) and stored (B) sample and LPE 18:0 at m/z 502. 295 (±0,025 m/z) for fresh (C) and stored (D) sample.

Figure 7. Tandem MS spectra of the proton adducts ([M+H]+) of regioisomeric LPC 0:0/18:1 (A) and 18:1/0:0 (B) at m/z 522.325 and LPE 18:0 (C) with m/z 502.295.