Article Cite This: Anal. Chem. XXXX, XXX, XXX-XXX
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A Comprehensive High-Resolution Targeted Workflow for the Deep Profiling of Sphingolipids Bing Peng,† Susan T. Weintraub,‡ Cristina Coman,† Srigayatri Ponnaiyan,§ Rakesh Sharma,∥,⊥ Björn Tews,∥,⊥ Dominic Winter,§ and Robert Ahrends*,† †
Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., 44227 Dortmund, Germany Department of Biochemistry and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States § Institute for Biochemistry and Molecular Biology, University of Bonn, 53113 Bonn, Germany ∥ Schaller Research Group, University of Heidelberg and DKFZ, 69120 Heidelberg, Germany ⊥ Molecular Mechanisms of Tumor Invasion, DKFZ, 69120 Heidelberg, Germany ‡
S Supporting Information *
ABSTRACT: Sphingolipids make up a highly diverse group of biomolecules that not only are membrane components but also are involved in various cellular functions such as signaling and protein sorting. To obtain a quantitative view of the sphingolipidome, sensitive, accurate, and comprehensive methods are needed. Here, we present a targeted reversedphase liquid chromatography−high-resolution mass spectrometry-based workflow that significantly increases the accuracy of measured sphingolipids by resolving nearly isobaric and isobaric species; this is accomplished by a use of (i) an optimized extraction procedure, (ii) a segmented gradient, and (iii) parallel reaction monitoring of a sphingolipid specific fragmentation pattern. The workflow was benchmarked against an accepted sphingolipid model system, the RAW 264.7 cell line, and 61 sphingolipids were quantified over a dynamic range of 7 orders of magnitude, with detection limits in the low femtomole per milligram of protein level, making this workflow an extremely versatile tool for high-throughput sphingolipidomics.
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Sphingolipids make up a highly diverse group of lipid species containing a sphingoid base backbone assembled out of a linear alkane and a 1,3-dihydroxy-2-amino headgroup. Sphingolipids as a class are structurally very heterogeneous as a consequence of the combination of various headgroup species, different numbers of hydroxyl groups, and the variation of the fatty acyl chains. The high degree of diversity, the wide dynamic range of sphingolipid concentrations in tissues and cells, interference with detection due to the presence of abundant phospholipids, and the existence of many isobaric and nearly isobaric sphingolipid species (mass difference of 17000 and a mass accuracy better than 57 ppm. However, even though the two nearly isobars can be clearly identified in MS1, they fragment together because of the 0.5 Da isolation window during precursor ion selection in PRM. Because there are multiple product ions in the majority of MS2 spectra of sphingolipids (Table S4 and Figure S5), the use of fragmentation patterns in conjunction with an accurate mass is the most effective way to accurately identify a sphingolipid species (Figure 5B). SRM has been the method of choice in sphingolipid analyses, but use of SRM can be problematic because the full MS2 profile is not acquired. In particular, if only the transition generating the LCB is monitored on a triplequadrupole mass spectrometer, it is not possible to detect the presence of nearly isobaric sphingolipids (e.g., HexCer d18:1/ 16:0 and CerP d18:1/22:1 that contain the same LCB fragment). This can be remedied through the use of PRM. The complete fragmentation pattern (Figures S5 and S6) can be utilized for confident identification within an individual sphingolipid class, supporting the distinction of isobaric precursors such as Cer d18:1/22:0 and Cer d18:0/22:1 (Figure E
DOI: 10.1021/acs.analchem.7b03576 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
Figure 5. Current challenges in sphingolipid analysis. (A) In challenge I, nearly isobaric species from low-resolution- and mass accuracy-derived data, e.g., from triple-quadrupole MS, are not always sufficient to resolve closely related species. (B) In challenge II, near isobars are sharing fragment ions. The chemical formulas (C40H83ON2P3, C43H76O5N2, C44H76O6, and C43H77O3N2P) are examples that also fit the detected precursor masses within a 5 ppm mass error. Collecting multiple fragments rather to rely on a single diagnostic fragment reduces false positive identification. Liquid chromatographic separation is required. (C) In challenge III, isobars overlap with isotopes. The PRM experiment via high-resolution MS showed disagreement with SRM via triple-quadrupole MS. The PRM overcomes SRM limitation by providing full MS2 spectra with multiple fragments with high resolution and mass accuracy to calculate transition intensity ratios (QIRs) by peak areas (a and b). (D) In challenge IV, isobaric species are at the precursor and fragment level. This challenge can be overcome only with the best possible resolution at LC, a full scan, and a PRM approach. Abbreviations: M, exact mass; EIC, extracted ion chromatogram.
Figure 6. KLA-induced autophagy in RAW 264.7 cells. (A) The unmodified and lipidated version of LC3 (LC3-II), as autophagy protein markers, is detected by Western blot. Tubulin staining serves as a loading control for the LC3-II protein. (B) Fold change in the relative amount of LC3-II in control and KLA-treated cells (based on densitometric quantification of three biological replicates). (C) Selected regulated sphingolipid species. All sphingolipids are listed in Figure S9. All experiments were performed in biological triplicate. *p < 0.05. F
DOI: 10.1021/acs.analchem.7b03576 Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
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forms, cytosolic LC3-I and membrane-bound LC3-II.35 LC3-II is a lipidated form of LC3 that has been shown to be an autophagosomal marker in mammals, providing evidence that autophagy is induced after KLA treatment.33 We monitored lipidated LC3-I and LC3-II by Western blot after KLA treatment and used tubulin as a loading control.36 Our results are in good agreement with earlier studies that observed an increase in the level of LC3-II (Figure 6B) and an increase in the LC3-II:LC3-I ratio between treatment and control (Figure 6A). This indicates induction of autophagosome formation in KLA-treated RAW264.7 cells. We found that the levels of most of the Cer species were increased after KLA treatment relative to SM (Figure 6C and Figure S9). Through use of our workflow, we identified more sphingolipid species with highquality spectra and high confidence than in previous reports.12,18 We were able to detect and quantify 50% more Cer species than earlier studies did.12,18 Cer levels were also altered after KLA treatment (Figures 6C and 7). The number
CONCLUSIONS A RPLC/HRMS-based workflow has been developed that significantly increases the accuracy in sphingolipidomics by resolving isobaric and nearly isobaric species through use of an efficient MTBE-based extraction approach that includes alkaline hydrolysis that removes interfering lipids, a segmented linear gradient that has been tailored for sphingolipids and effectively separates closely eluting sphingolipid species, and an MS strategy that includes HRMS survey scans and PRM MS detection. Our workflow provides enhancements in sample preparation and analysis that yield a higher level of confidence for sphingolipid identification and quantification. Application of the workflow to analysis of sphingolipids in RAW 264.7 cells demonstrates its utility for samples of biological origin.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03576. Tables S1−S5, Figures S1−S9, and additional information as mentioned in the text (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Otto-Hahn-Str .6b, 44227 Dortmund, Germany. Phone: +49231-1392-4173. Fax: +49-231-1392-4173. E-mail: robert.
[email protected]. ORCID
Cristina Coman: 0000-0002-3771-2410 Dominic Winter: 0000-0001-6788-6641 Robert Ahrends: 0000-0003-0232-3375
Figure 7. Comparison of identified lipid species in the literature and this work. Black bars represent data from the study presented here and light gray and dark gray bars data from earlier studies. We were able to detect and quantify 50% more Cer species than earlier studies did. The number of HexCer, SM, and CerP species identified by our workflow is lower than in other reports. However, there is a high level of confidence associated with our identifications, because they are based on high-resolution/high-mass accuracy precursor and fragment ion data in addition to complete fragmentation information at the MS2 level.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin, and the Bundesministerium für Bildung und Forschung and by the BMBF de.NBI program (code 031L0108A). The authors thank Dr. Christian Hellmuth for valuable comments on the manuscript.
of HexCer, SM, and CerP species identified by our workflow is technically lower than in other reports. However, there is a high level of confidence associated with our identifications, because they are based on high-resolution/high-mass accuracy precursor data and the complete tandem mass spectral pattern. In contrast, only the LCB (i.e., one fragment) that is shared among many sphingolipid species was used for sphingolipid identification in the referenced papers. Use of the sphingolipid fragmentation pattern in combination with high-resolution/ high-mass accuracy precursor measurements increases the reliability substantially, because many false positive lipids are removed from consideration. In total, we identified and quantified 61 sphingolipid species within a dynamic range of 7 orders of magnitude with detection ranging to the low femtomole per milligram protein level. In agreement with earlier studies of the same cell type,18,37 sulfatides were not identified after KLA treatment.
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DOI: 10.1021/acs.analchem.7b03576 Anal. Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.analchem.7b03576 Anal. Chem. XXXX, XXX, XXX−XXX
