LC-MS based sphingolipidomic study on A549 human lung adenocarcinoma cell line and its taxol-resistant strain

The LIPID MAPS internal standard cocktail (internal standards mixture II, 25 μM each of 9 compounds in ethanol, catalog LM-6005) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). It was composed of uncommon SPLs which include: 17-carbon chain length sphingoid base analogs C17-sphingosine [So (d17:1)], C17-sphinganine [Sa (d17:0)], C17-sphingosine-1-phosphate [S1P (d17:1)], C17-sphinganine-1-phosphate [Sa1P (d17:0)], the C12-fatty acid analogs of the more complex SPLs C12-Ceramide [Cer (d18:1/12:0)], C12-ceramide-1-phosphate [C1P (d18:1/12:0)], C12-sphingomyelin [SM (d18:1/12:0)], C12-glucosylceramide [GlcCer (d18:1/12:0)], and C12-lactosylceramide [LacCer (d18:1/12:0)].

Acetic acid (CH₃COOH, MS grade), formic acid (HCOOH, MS grade), ammonium acetate (NH4OAc, ACS grade) and potassium hydroxide (KOH, ACS grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HPLC grade chloroform (CHCl₃), isopropanol (IPA), as well as methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Fetal Bovine Serum (FBS), Penicillin-Streptomycin (PS) were obtained from Gibco, New Zealand. Sodium dodecyl sulfate (SDS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were acquired from Acros, USA. Ultrapure water (18.2 MΩ) was supplied with a Milli-Q system (Millipore, MA, USA).

A549 human lung adenocarcinoma cell line (Cat.No. KG007) and its taxol-resistant strain (A549T, Cat.No. KG124) were obtained from KeyGen Biotech Co., Ltd. (Nanjing, China). A549 was cultured in DMEM supplemented with 10% FBS and 1% PS in a humidified 5% CO₂ atmosphere at 37 °C. A549T was cultured in RPMI 1640 medium supplemented with solution consisted of 10% FBS, 1% PS and 200 ng/mL taxol in a humidified 5% CO₂ atmosphere at 37 °C. For lipid analysis, A549 and A549T cells were respectively seeded into 6-well plates at the density of 1.5 × 10⁵ cells/well and incubated for 48 h. Lipids were extracted from the cells, when they were grown to 80% confluence. After rinsed twice by ice-cold PBS, the cells were scraped into a borosilicate glass tube, in which 0.5 mL of MeOH, 0.25 mL of CHCl₃ and 10 μL of 2.5 μM internal standards cocktail were added. The extract procedure was carried out by incubation at 48 °C for 12 h after sonicated at ambient temperature for 30 s. After 75 μL of KOH in MeOH (1 M) was added, the mixture was placed into a shaking incubator at 37 °C for 2 h. Acetic acid was used to neutralize the mixture before the typical four-step extraction was carried out for the preparation of SPLs. Further details for extracting SPLs and sample preparation were the same as previously described [11]. MTT assay was employed to evaluate the sensitivity of A549 and A549T cells to taxol. The IC₅₀s were 67.72 nM and 124.7 μM, respectively corresponding to A549 and A549T, showing almost 2000-fold difference in taxol sensitivity between these two cell lines.

Sphingolipid analysis was performed by using our developed LC-MS method with minor optimization, just as described previously [10, 11]. Chromatographic separation was achieved by using an Agilent 1290 UHPLC system, and it was interfaced with an Agilent ultrahigh definition 6550 Q-TOF mass spectrometer and an Agilent 6460 triple-quadrupole mass spectrometer respectively for qualitative- and quantitative-analysis. The acquisition and data analysis were operated by using Agilent MassHunter Workstation Software.

Based on the Agilent Personal Compound Database and Library (PCDL) software and LIPID MAPS Lipidomics Gateway, a personal database has been established with the latest update of 32,622 SPLs until August 06 2016. The screening and identification of SPLs were carried out by searching against it.

In qualitative research, the sphingolipidomic approach was applied by analyzing QC samples equally pooled by A549 and A549T. In quantitative research, A549 cells (models, n = 10) and A549T cells (models, n = 10), as well as QC samples (n = 5), were analyzed in parallel. Multivariate statistical analysis, including principle component analysis (PCA) and partial least squares to latent structure-discriminant analysis (PLS-DA) methods, were performed to examine significant differences between A549 and A549T, using SIMCA-P+ software version 14.0 (Umetrics, Umea, Sweden). Variable Importance in the Project (VIP) value in PLS-DA model was used for selecting and identifying biomarkers. The altered SPL with a VIP value larger than 1.00 was considered as a biomarker.

QC samples were analyzed repeatedly to achieve comprehensive profiling of SPLs in A549 and A549T. In various subclasses of SPLs, the [M + H]⁺ ions exhibits highest intensities in positive ion mode. Totally 114 SPLs have been identified in the QC samples, among which Cer (d18:2/26:2), DHCer (d18:0/24:2), phytosphingosine (PTSo) t19:2, and PTSo t16:1 were new SPLs. Notably, 4 pairs of isobaric species (A₁-A₄ vs a₁-a₄) and 21 pairs of isomeric species (B₁-B₂₁ vs b₁-b₂₁) were clearly distinguished in this study. Respective qualitative test of A549 and A549T revealed that they share all the same species of SPLs. The full identification result was listed in Table 1.

Interpretation of high resolution MS and MS/MS spectra of each identified ion, as well as searching against the latest database, allowed for the accurate identification of SPLs. For instance, isobaric lipids could be differentiated by the high-resolution mass spectrometry-based approaches. Two peaks yield m/z 316 ions, with accurate mass acquired by Q-TOF, m/z 316.3202 at 6.532 min and m/z 316.2850 at 6.874 min correspond to [C₁₉H₄₁NO₂ + H]⁺ and [C₁₈H₃₇NO₃ + H]⁺ respectively, facilitating assignment of sphinganine (Sa) d19:0 and phytosphingosine (PTSo) t18:1. Further fragmentation in MS/MS confirmed the identification, a consecutive loss of 3 hydroxy groups can be observed in the latter case, which is the characteristic cleavage of PTSo (Fig. 1).

A more realistic interference in the identification of SPLs is the isomeric species that have same number of atoms of each element, thus MS/MS fragment data with the assistance of optimized separation are essential for distinguishing the isomers. Take SM (d18:1/22:1) and SM (d18:2/22:0) as example, there are 2 peaks corresponding to m/z 785.65 in extracted ion chromatogram of TOF MS. In accurate MS/MS data acquired by Q-TOF, two characteristic fragments (264.3 & 262.3) respectively corresponding to the sphingoid base chain of SM (d18:1/22:1) and SM (d18:2/22:0) were observed (Fig. 2). The targeted ion pairs as well as complete chromatographic separation make the accurate MRM quantification of isomers possible.

Ceramides are prone to fragment into product ions corresponding to the sphingoid base backbone (e.g. m/z 262.25, 264.27, 266.28). In A549 QC samples, 29 Cers, including 20 dehydroceramides, 8 dihydroceramides (DHCers) and 1 phytoceramide (PTCer), were identified by comparing the MS information and retention time with those of SPLs in our previous study [10, 11]. Most Cers detected in the samples were with a d18:1 sphingoid backbone and the carbon number of N-acyl side chain varied from 14 to 26. A new dihydroceramide DHCer (d18:0/24:2), and Cer (d18:2/26:2), a dehydroceramide with high degree of unsaturation and long N-acyl chain, have been characterized for the first time to the best of our knowledge.

SM is the most multitudinous subclass of SPLs in A549 and A549T. Based on the exact mass in TOF MS and characteristic product ions obtained by Q-TOF MS/MS, a total of 56 SMs, including 38 dehydrosphingomyelins and 18 dihydrosphingomyelins (DHSMs), were unambiguously identified. All these SMs were characterized with a C18 sphingoid base chain, among which d18:1 type takes the largest proportion. In the N-acyl side chain, the number of carbon ranged between 14 and 26, with an unsaturation degree up to 5. Notably, all the DHSMs with 21 or less carbons in the N-acyl chain are fully saturated, while the others (with more than 21 carbons in the N-acyl chain) can be detected together with their corresponding de-hydrogen form. Three highly unsaturated SMs (total unsaturation degree no less than 4) including SM (d18:1/24:3), SM (d18:2/24:2) and SM (d18:2/24:3), have been detected in the QC sample of A549 & A549T cells.

Hexose-linked glycoceramide including galactosylceramide (GalCer) and glucosylceramide (GluCer) were represented as HexCer. All the 6 HexCers and 6 LacCers were found with d18:1 sphingoid base backbone. Only one HexCer with N-acyl chain in odd carbon number, HexCer (d18:1/23:0) was identified in A549. Notably, among all the HexCers and LacCers, only d18:1/24:1 species were identified as glycoceramides with unsaturated N-acyl fatty chain.

Seventeen sphingoid bases as well as the analogs were also successfully identified. The carbon number ranging from 14 to 19 and the degree of unsaturation falls between 0 and 2. Two PTSo with 3 hydroxyl groups, PTSo t19:2 and PTSo t16:1, have been discovered for the first time.

MRM mode of UHPLC-QQQ MS could provide accurate and sensitive approach under a wide range for quantitative analysis of SPLs. As the accuracy of triple-quadruple is about 0.1 Da, the quantification of SPLs cannot be accurately achieved merely with a QQQ analyzer, especially when suffering the isotopic interferences. Every unsaturated SPL could be recognized as an isotope of another one with the same characteristic backbone but less degree of unsaturation. For instance, if the LC separation is incomplete, the content of Cer (d18:1/24:0) will be artificially high due to the interference of Cer (d18:1/24:1) (Fig. 3). In this study, based on UHPLC complete separation and Q-TOF comprehensive profiling, accurate quantification was accomplished by eliminating the isotopic interference. By using the UHPLC-QQQ MS method with the optimized MRM parameters, a total of 75 species out of 114 identified SPLs were quantified in A549 and A549T cells, respectively. The amounts of these SPLs were quantified by comparing with the foregoing mentioned ISs.

The quantitative results indicated that SMs account for the majority of all the SPLs in A549 and A549T, among which SMs with C16/C18/C22/C24 N-acyl side chain took the largest proportion of the total content. SMs with d18:1 sphingoid backbone are the most dominant species, which take 27 out of all the 41 quantified SMs (Fig. 4). For some SMs with high unsaturation degree or long N-acyl chain, the content is extremely low which cannot reach the limit of quantitation (LOQ). Figure 5 shows quantification data of 17 Cers. In general, the amounts of various Cers are significantly higher in A549 rather than those in A549T. Similar to SM, d18:1 Cers with C16/C18/C22/C24 N-acyl side chain showed relative high levels in both A549 and A549T, which take most proportion of Cer. LacCers and HexCers were only found with d18:1 sphingoid base backbone. All the 6 LacCers showed higher intensity in A549T than that in A549. But HexCer showed a species-dependent trend, HexCer d18:1/16:0, HexCer d18:1/22:0 and HexCer d18:1/23:0 increased in A549T, while HexCer d18:1/24:0, HexCer d18:1/24:1 and HexCer d18:1/26:0 decreased (Fig. 6). The overall content of sphingoid bases was similar in both cell types, Sa d16:0 was found with the highest intensity (Fig. 7). The relative abundance of each SPL varied greatly, but SPLs with N-acyl chain length of C16 and C24, respectively, are the most abundant species within each subclass.

PCA was used for the overview of SPL dataset and the spotting of outliers, and thereby pick out trends of grouping or separation. It was performed to visualize general clustering among A549, A549T and QC groups [R²X (cum) = 0.874, Q² (cum) = 0.845; Fig. 8a]. Supervised PLS-DA was used to further study the differences between A549 and A549T and to select potential biomarkers. In PLS-DA, the result of model showed the performance statistics of R²X (cum) = 0.880, R²Y (cum) = 0.999 with an excellent prediction parameter Q² (cum) = 0.998, and the score plot showed good visual separation between A549 and A549T groups as well (Fig. 8b). A total of 57 potential biomarkers were identified according to scattering-plot and the VIP value (Table 2), among which most of them are SM and Cers. SM (d18:0/18:0) showed the largest decline in A549T, the content in decreased from 17.0 to 0.10 pmol/(5 × 10⁵cells), that markedly contributes to the classification.