Improved Spatial Resolution of MALDI Imaging of Lipids in the Brain by Alkylated Derivatives of 2,5-Dihydroxybenzoic Acid

The cell/tissue lipidome comprises over 10,000 individual species of highly diverse molecules.[1, 2] The identification and detailed structural characterization of lipids have been markedly accelerated by the development of soft ionization techniques for mass spectrometry (MS) combined with chromatography.[1, 2] Recently, MS has advanced into the field of imaging to enable the spatial cataloging of lipids in cellular compartments (reviewed in [3, 4]). The most commonly employed mass spectrometry imaging (MSI) techniques for lipids are secondary ion mass spectrometry (SIMS), desorption electrospray ionization (DESI), and matrix-assisted laser desorption/ionization (MALDI). These techniques have been successfully applied to depict the spatial distribution of endogenous and exogenous molecules, as well as for revealing correlations of the homeostasis of low molecular mass compounds and proteins with disease occurrence.[3–6] In MALDI-MSI, tissue slices are coated with a matrix that is subsequently ablated by absorbing energy from a laser beam. This desorption forms a plume that also contains intact analyte molecules that were in close proximity to the matrix. The analytes either retain pre-formed charges or engage in charge-transfer reactions in the gas phase.[7, 8] This analysis is repeated at discrete locations on the tissue section, resulting in a “molecular fingerprint” that correlates analyte abundance with spatial location.[3, 4] An intrinsic limitation of MALDI-MSI of lipids is the irregular crystallization of matrices on the analyzed tissue surface. Variability in the matrix-to-analyte ratio affects ion intensity,[9, 10] an issue that becomes a limiting factor in MALDI-MSI at high lateral resolution.[11] Careful optimization of sample preparation and matrix deposition methods are necessary to achieve the best resolution possible with MALDI imaging.[12–14] 2,5-dihydroxybenzoic acid (DHB) is a widely used matrix for MALDI-MSI of biological molecules, including the mapping of (phospho)lipids.[3, 5] Either sublimation of DHB or dry-coating of DHB crystals through a sieve can give a coating of small crystals over the tissue surface[15, 16]. Spray coating can be optimized to deliver a matrix layer that is fine enough to image individual HeLa cells.[17] As a general rule, smaller matrix crystals and stronger absorbance by the matrix at the laser wavelength yields a more efficient MALDI process, whereby optimal MALDI-MSI is achieved through maximum contact between analytes and matrix molecules.[18] DHB is a weak acid (pKa = 2.95) that facilitates MALDI-MS detection of lipids that are prone to protonation.[19] For enhanced protonation of analytes, trifluoroacetic acid (TFA; pKa = 0.23) is often used as an additive to DHB. However, recent MALDI-MS studies demonstrated that the combination of DHB/TFA can cause significant hydrolysis of phospholipids.[20] DHB exhibits a UV spectrum with a maximum at 355 nm, making it a suitable matrix for analyses with MALDI mass spectrometers with N2 lasers centered at 337 nm. At the optimal analyte-to-matrix ratio, the UV absorbance maximum of DHB should not deviate significantly from the laser wavelength as this would decrease the energy transfer from the laser to the analytes.[18] Optimizing the absorbance of DHB by modifying the aromatic ring with various substituents gives an improved energy transfer.[21] Modifying the physical properties of the matrix will also give improved MALDI-MS performance. Detergents with acid-cleavable alkyl chains have been used to improve the tryptic digestion efficiency of membrane proteins without interfering with MALDI-MS.[13] Matrices with acid-cleavable alkyl chains resulted in a functional detergent-matrix compound that gave greatly improved signals for cell lysates and membrane proteins and generated the unmodified matrix itself upon sample preparation.[14] Noticeably, after cleavage of the alkyl chain, the crystallization properties of the resulting matrix were unchanged from that of the non-alkylated matrix. DHB with various length alkyl chains, when added to another matrix, gave improved signals of up to two orders of magnitude for hydrophobic peptides.[9] The crystallization properties of this mixture of alkylated and non-alkylated matrices were different from that of the non-alkylated matrix alone, and resulted in different distributions of hydrophobic peptides within the sample spot. In the present study, we have carried out experiments to test whether the introduction of linear hydrocarbon chains in DHB yields matrices with improved analytical characteristics for MALDI-MSI of phospholipids in brain tissue. Targeted synthesis of DHB-CnH2n+1 (C6H13, C9H19 and C12H25) was carried out following the hypothesis that alkyl derivatives of DHB form smaller crystals, which is a pre-requisite for higher spatial resolution, and that incorporation of the alkyl chains of DHB-CnH2n+1 into phospholipid bilayers during matrix deposition on tissue slices will provide better matrix-to-analyte contact.[22, 23]