Abstract
Unequivocal assignment of phospholipid peaks in complex mixtures is difficult if only the m/z values but no tandem mass spectrometry (MS/MS) data are available. This is usually the case for matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) MS imaging experiments and the analysis has normally to be performed without prior separation. Another problem might be the often matrix-induced loss of one methyl group in phosphatidylcholine (PC) species, which makes them detectable as negative ions becoming isomers of some phosphatidylethanolamines (PEs). Selected lipid mixtures of known compositions were investigated by negative ion MALDI-TOF MS and various imaging experiments. In addition to common matrices such as 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), different binary matrices, including 2,5-dihydroxyacetophenone (2,5-DHAP) as matrix additive to DHB, were tested to probe their performance in both ionization modes. Beside artificial PC and PE mixtures of known compositions, egg yolk and liver extracts as well as cryosections from liver and pancreas tissue were selected as biologically relevant systems. The majority of the binary MALDI matrices used here leads to the loss of a methyl group from PC in the negative ion mode, which makes the clear identification of PE species ambiguous. However, this problem does not apply if a mixture of DHB and 2,5-DHAP is used. Therefore, the application of DHB/2,5-DHAP as matrix is a simple method to unequivocally identify PEs even in complex mixtures and tissue sections as negative ions and without the necessity to separate the individual lipid classes prior to MS detection.
Similar content being viewed by others
References
Wenk MR. The emerging field of lipidomics. Nat Rev Drug Discov. 2005;4:594–610.
Wurtman R. Biomarkers in the diagnosis and management of Alzheimer’s disease. Metabolism. 2015;64:47–50.
Fuchs B. Mass spectrometry and inflammation—MS methods to study oxidation and enzyme-induced changes of phospholipids. Anal Bioanal Chem. 2014;406:1291–306.
Bandu R, Mok HJ, Kim KP. Phospholipids as cancer biomarkers: mass spectrometry-based analysis. Mass Spectrom Rev. 2016; in press. https://doi.org/10.1002/mas.21510.
Fuchs B, Süss R, Schiller J. An update of MALDI-TOF mass spectrometry in lipid research. Prog Lipid Res. 2010;49:450–75.
Boellner S, Becker KF. Recent progress in protein profiling of clinical tissues for next-generation molecular diagnostics. Expert Rev Mol Diagn. 2015;15:1277–92.
Ellis SR, Brown SH, in Het Panhuis M, Blanksby SJ, Mitchell TW. Surface analysis of lipids by mass spectrometry: more than just imaging. Prog Lipid Res. 2013;52:329–53.
Aichler M, Walch A. MALDI imaging mass spectrometry: current frontiers and perspectives in pathology research and practice. Lab Investig. 2015;95:422–31.
Baker TC, Han J, Borchers CH. Recent advancements in matrix-assisted laser desorption/ionization mass spectrometry imaging. Curr Opin Biotechnol. 2017;43:62–9.
Luxembourg SL, McDonnell LA, Duursma MC, Guo X, Heeren RM. Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry. Anal Chem. 2003;75:2333–41.
Sun G, Yang K, Zhao Z, Guan S, Han X, Gross RW. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions. Anal Chem. 2008;80:7576–85.
Teuber K, Schiller J, Fuchs B, Karas M, Jaskolla JW. Significant sensitivity improvements by matrix optimization: a MALDI-TOF mass spectrometric study of lipids from hen egg yolk. Chem Phys Lipids. 2010;163:552–60.
Fuchs B, Bischoff A, Süss R, Teuber K, Schürenberg M, Suckau D, et al. Phosphatidylcholines and -ethanolamines can be easily mistaken in phospholipid mixtures: a negative ion MALDI-TOF MS study with 9-aminoacridine as matrix and egg yolk as selected example. Anal Bioanal Chem. 2009;395:2479–87.
Dertinger JJ, Walker AV. Towards the rational design of ionic liquid matrices for secondary ion mass spectrometry: role of the anion. J Am Soc Mass Spectrom. 2013;24:1288–95.
Meriaux C, Franck J, Wisztorski M, Salzet M, Fournier I. Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids. J Proteome. 2010;73:1204–18.
Mank M, Stahl B, Boehm G. 2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules. Anal Chem. 2004;76:2938–50.
Karas M, Ehring H, Nordhoff E, Stahl B, Strupat K, Hillenkamp F, et al. Matrix-assisted laser desorption/ionization mass spectrometry with additives to 2,5-dihydroxybenzoic acid. Rapid Commun Mass Spectrom. 1993;28:1476–81.
Shanta SR, Kim TY, Hong JH, Lee JH, Shin CY, Kim KH, et al. A new combination MALDI matrix for small molecule analysis: application to imaging mass spectrometry for drugs and metabolites. Analyst. 2012;137:5757–62.
Shanta SR, Zhou LH, Park YS, Kim YH, Kim Y, Kim KP. Binary matrix for MALDI imaging mass spectrometry of phospholipids in both ion modes. Anal Chem. 2011;83:1252–9.
Calvano CD, Monopoli A, Ditaranto N, Palmisano F. 1,8-Bis(dimethylamino)naphthalene/9-aminoacridine: a new binary matrix for lipid fingerprinting of intact bacteria by matrix assisted laser desorption ionization mass spectrometry. Anal Chim Acta. 2013;798:56–63.
Wang J, Wang C, Han X. Enhanced coverage of lipid analysis and imaging by matrix-assisted laser desorption/ionization mass spectrometry via a strategy with an optimized mixture of matrices. Anal Chim Acta. 2018;1000:155–62.
Hayasaka T, Goto-Inoue N, Masaki N, Ikegami K, Setou M. Application of 2,5-dihydroxyacetophenone with sublimation provides efficient ionization of lipid species by atmospheric pressure matrix-assisted laser desorption/ionization imaging mass spectrometry. Surf Interface Anal. 2014;46:1219–22.
Fülöp A, Porada MB, Marsching C, Blott H, Meyer B, Tambe S, et al. 4-Phenyl-α-cyanocinnamic acid amide: screening for a negative ion matrix for MALDI-MS imaging of multiple lipid classes. Anal Chem. 2013;85:9156–63.
Schiller J, Arnhold J, Benard S, Müller M, Reichl S, Arnold K. Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: a methodological approach. Anal Biochem. 1999;267:46–56.
Vidová V, Pól J, Volny M, Novák P, Havlícek V, Wiedmer SK, et al. Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry. J Lipid Res. 2010;51:2295–302.
AlMasoud N, Correa E, Trivedi DK, Goodacre R. Fractional factorial design of MALDI-TOF-MS sample preparations for the optimized detection of phospholipids and acylglycerols. Anal Chem. 2016;88:6301–8.
Astigarraga E, Barreda-Gómez G, Lombardero L, Fresnedo O, Castaño F, Giralt MT, et al. Profiling and imaging of lipids on brain and liver tissue by matrix-assisted laser desorption/ ionization mass spectrometry using 2-mercaptobenzothiazole as a matrix. Anal Chem. 2008;80:9105–14.
Calvano CD, Ventura G, Cataldi TR, Palmisano F. Improvement of chlorophyll identification in foodstuffs by MALDI ToF/ToF mass spectrometry using 1,5-diaminonaphthalene electron transfer secondary reaction matrix. Anal Bioanal Chem. 2015;407:6369–79.
Estrada R, Yappert MC. Alternative approaches for the detection of various phospholipid classes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Mass Spectrom. 2004;39:412–22.
Wang HY, Jackson SN, Woods AS. Direct MALDI-MS analysis of cardiolipin from rat organs sections. J Am Soc Mass Spectrom. 2007;18:567–77.
Blight EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–7.
Eibisch M, Fuchs B, Schiller J, Süß R, Teuber K. Analysis of phospholipid mixtures from biological tissues by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS): a laboratory experiment. J Chem Educat. 2011;88:503–7.
Popkova Y, Schiller J. Addition of CsCl reduces ion suppression effects in the matrix-assisted laser desorption/ionization mass spectra of triacylglycerol/phosphatidylcholine mixtures and adipose tissue extracts. Rapid Commun Mass Spectrom. 2017;31:411–8.
Schiller J, Süss R, Petković M, Zschörnig O, Arnold K. Negative-ion matrix-assisted laser desorption and ionization time-of-flight mass spectra of complex phospholipid mixtures in the presence of phosphatidylcholine: a cautionary note on peak assignment. Anal Biochem. 2002;309:311–4.
Thomas A, Charbonneau JL, Fournaise E, Chaurand P. Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition. Anal Chem. 2012;84:2048–54.
Schiller J, Süss R, Fuchs B, Müller M, Petković M, Zschörnig O, et al. The suitability of different DHB isomers as matrices for the MALDI-TOF MS analysis of phospholipids: which isomer for what purpose? Eur Biophys J. 2007;36:517–27.
Fuchs B, Schiller J, Süss R, Schürenberg M, Suckau D. A direct and simple method of coupling matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to thin-layer chromatography (TLC) for the analysis of phospholipids from egg yolk. Anal Bioanal Chem. 2007;389:827–34.
Hinsenkamp I, Schulz S, Roscher M, Suhr AM, Meyer B, Munteanu B, et al. Inhibition of rho-associated kinase 1/2 attenuates tumor growth in murine gastric cancer. Neoplasia. 2016;18:500–11.
Asimakopoulou A, Fülöp A, Borkham-Kamphorst E, van de Leur E, Gassler N, Berger T, et al. Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis. Biochim Biophys Acta. 1863;2017:2093–110.
Acknowledgements
This study was supported by the German Research Council (DFG Schi 476/12-2, DFG Schi 476/16-1, and SFB 1052/Z3). We would also like to thank all our colleagues who helped us perform related experiments. We are particularly indebted to Bruker Daltonik GmbH (Bremen, Germany) and Merck Millipore (Darmstadt, Germany) for the continuous and generous support.
Funding
C.H. thanks the Deutsche Forschungsgemeinschaft (DFG) for the funding of a SolariX FTICR MS (INST 874/7-1 FUGG).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
Electronic supplementary material
ESM 1
(PDF 1292 kb)
Rights and permissions
About this article
Cite this article
Schröter, J., Fülöp, A., Hopf, C. et al. The combination of 2,5-dihydroxybenzoic acid and 2,5-dihydroxyacetophenone matrices for unequivocal assignment of phosphatidylethanolamine species in complex mixtures. Anal Bioanal Chem 410, 2437–2447 (2018). https://doi.org/10.1007/s00216-018-0926-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-018-0926-9