4‑Phenyl-α-cyanocinnamic Acid Amide: Screening for a Negative Ion Matrix for MALDI-MS Imaging of Multiple Lipid Classes Annabelle Fü lö p, †,‡,§ Martina B. Porada, †,∥ Christian Marsching, †,‡,§,⊥ Henning Blott, †,∥ Bjö rn Meyer, †,‡,§ Suparna Tambe, †,∥ Roger Sandhoff, †,‡,⊥ Hans-Dieter Junker, †,∥ and Carsten Hopf* ,†,‡,§ † Applied Research Center in Biomedical Mass Spectrometry (ABIMAS), ‡ Instrumental Analysis and Bioanalysis, Mannheim University of Applied Sciences, Paul-Wittsack-Strasse 10, 68163 Mannheim, Germany § Institute of Medical Technology, University of Heidelberg and Mannheim University of Applied Sciences, Paul-Wittsack-Strasse 10, 68163 Mannheim, Germany ∥ Organic Chemistry, Aalen University of Applied Sciences, Beethovenstrasse 1, 73430 Aalen, Germany ⊥ Lipid Pathobiochemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany * S Supporting Information ABSTRACT: Matrix-assisted laser desorption/ionization imaging mass spec- trometry (MALDI-IMS) has become a method of choice in lipid analysis, as it provides localization information for defined lipids that is not readily accessible with nonmass spectrometric methods. Most current MALDI matrices have been found empirically. Nevertheless, preferential matrix properties for many analyte classes are poorly understood and may differ between lipid classes. We used rational matrix design and semiautomated screening for the discovery of new matrices suitable for MALDI-IMS of lipids. Utilizing Smartbeam- and nitrogen lasers for MALDI, we systematically compared doubly substituted α- cyanocinnamic acid derivatives (R 1 -CCA-R 2 ) with respect to their ability to serve as negative ion matrix for various brain lipids. We identified 4-phenyl-α- cyanocinnamic acid amide (Ph-CCA-NH 2 ) as a novel negative ion matrix that enables analysis and imaging of various lipid classes by MALDI-MS. We demonstrate that Ph-CCA-NH 2 displays superior sensitivity and reproducibility compared to matrices commonly employed for lipids. A relatively small number of background peaks and good matrix suppression effect could make Ph-CCA-NH 2 a widely applicable tool for lipid analysis. L ipids are major building blocks of biological membranes, and they play key roles in signal transduction pathways 1,2 and mechanisms of disease. 3,4 Because of these important functions, the field of lipid analysis and lipidomics has made considerable progress in recent years. 5,6 Matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS) 7 in general and MALDI imaging MS (MALDI-IMS) 8,9 in particular, which provides information about the location of different lipid species, have become methods of choice for lipid studies. 10−14 In a typical MALDI- IMS experiment, a thin tissue cryosection is mounted onto a suitable target and covered with a chemical matrix solution that extracts analytes of interest from the underlying tissue. Some matrices limit the reproducibility and the spatial resolution of MALDI imaging experiments because of large or inhomoge- neous crystal formation on the tissue slices. In addition, inhomogeneous cocrystallization of analytes with matrices can lead to the existence of so-called hot spots 15 on the sample, which causes quantitative errors and complicates automated measurement. Therefore, the role of the matrix is to cocrystallize with analytes and to absorb laser energy, in order to promote desorption/ionization. The challenges in imaging stem from the wide range of lipid concentrations and the very different ionization efficiencies of various lipid species. It is therefore necessary to choose a matrix that is either selective for a defined class of lipids 16,17 or that facilitates efficient ionization of a maximum number of lipid classes independent of their distinct physicochemical properties. In addition, a suitable matrix should possess a high absorbance at the emission wavelength of the laser and a low matrix background. 18,19 The presence of alkali metal ions in tissue slices leads to formation of multiple adducts for each lipid species in MALDI images in positive ion mode and thus complicates their interpretation. In contrast, in negative ion spectra the exclusive presence of [M − H] − ion signals formed from a large variety of lipids facilitates the analysis. The most frequently used matrices for lipid MALDI-IMS in negative ion mode are 2,5-dihydroxybenzoic acid (DHB) 20 and 9-aminoacridine (9-AA). 17,21 However, DHB tends to form large crystals, which may cause molecular delocalization and poor spot-to-spot reproducibility, 22,23 and it exhibits only poor analyte sensitivities accompanied by strong background noise in Received: June 17, 2013 Accepted: August 28, 2013 Published: August 28, 2013 Article pubs.acs.org/ac © 2013 American Chemical Society 9156 dx.doi.org/10.1021/ac4018154 | Anal. Chem. 2013, 85, 9156−9163