Multimodal imaging of biological tissues using combined MALDI and NAPA-LDI mass spectrometry for enhanced molecular coverage.

Mass spectrometry imaging (MSI) is a powerful analytical technique that enables detection, discovery, and identification of multiple classes of biomolecules, while simultaneously mapping their spatial distributions within a sample (e.g., a section of biological tissue). The limitation in molecular coverage afforded by any single MSI platform has led to the development of multimodal approaches that incorporate two or more techniques to obtain greater chemical information. Matrix-assisted laser desorption ionization (MALDI) is a preeminent ionization technique for MSI applications because the wide range of available matrices allows some degree of enhancement with respect to the detection of particular molecular classes. Nonetheless, MALDI has a limited ability to detect and image several classes of molecules, e.g., neutral lipids, in complex samples. Laser desorption ionization from silicon nanopost arrays (NAPA-LDI or NAPA) has been shown to offer complementary coverage with respect to MALDI by providing improved detection of neutral lipids and some small metabolites. Here, we present a multimodal imaging method in which a single tissue section is consecutively imaged at low and high laser fluences, generating spectra that are characteristic of MALDI and NAPA ionization, respectively. The method is demonstrated to map the distributions of species amenable to detection by MALDI (e.g., phospholipids and intermediate-mass metabolites) and NAPA (e.g., neutral lipids such as triglycerides and hexosylceramides, and small metabolites) in mouse brain and lung tissue sections.

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays.

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix-assisted laser desorption ionization (MALDI) is recognized as the method-of-choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix-free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI-MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227-fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295-fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na+ . Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49-fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19-fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.

High Throughput Complementary Analysis and Quantitation of Metabolites by MALDI- and Silicon Nanopost Array-Laser Desorption/Ionization-Mass Spectrometry.

Silicon nanopost array (NAPA) structures have been shown to be effective substrates for laser desorption/ionization-mass spectrometry (LDI-MS) and have been used to analyze a variety of samples including peptides, metabolites, drugs, explosives, and intact cells, as well as to image lipids and metabolites in tissue sections. However, no direct comparison has yet been conducted between NAPA-MS and the most commonly used LDI-MS technique, matrix-assisted laser desorption/ionization (MALDI)-MS. In this work, we compare the utility of NAPA-MS to that of MALDI-MS using two common matrices for the analysis of metabolites in cellular extracts and human urine. Considerable complementarity of molecular coverage was observed between the two techniques. Of 178 total metabolites assigned from cellular extracts, 68 were uniquely detected by NAPA-MS and 62 were uniquely detected by MALDI-MS. NAPA-MS was found to provide enhanced coverage of low-molecular weight compounds such as amino acids, whereas MALDI afforded better detection of larger, labile compounds including nucleotides. In the case of urine, a sample largely devoid of higher-mass labile compounds, 88 compounds were uniquely detected by NAPA-MS and 13 by MALDI-MS. NAPA-MS also favored more extensive alkali metal cation adduction relative to MALDI-MS, with the [M + 2Na/K - H]+ species accounting for as much as 97% of the total metabolite ion signal in positive mode. The capability of NAPA-MS for targeted quantitation of endogenous metabolites in urine via addition of isotopically labeled standards was also examined. Both NAPA-MS and MALDI-MS provided quantitative results in good agreement with one another and the concentrations reported in the literature, as well as good sample-to-sample reproducibility (RSD < 10%).

Matrix-free mass spectrometry imaging of mouse brain tissue sections on silicon nanopost arrays.

Mass spectrometry imaging (MSI) is capable of detection and identification of diverse classes of compounds in brain tissue sections, whereas simultaneously mapping their spatial distributions. Given the vast array of chemical components present in neurological systems, as well as the innate diversity within molecular classes, MSI platforms capable of detecting a wide array of species are useful for achieving a more comprehensive understanding of their biological roles and significance. Currently, matrix-assisted laser desorption ionization (MALDI) is the method of choice for the molecular imaging of brain samples by mass spectrometry. However, nanostructured laser desorption ionization platforms, such as silicon nanopost arrays (NAPA), are emerging as alternative MSI techniques that can provide complementary insight into molecular distributions in the central nervous system. In this work, the molecular coverage of mouse brain lipids afforded by NAPA-MSI is compared to that of MALDI-MSI using two common MALDI matrices. In positive ion mode, MALDI spectra were dominated by phosphatidylcholines and phosphatidic acids. NAPA favored the ionization of phosphatidylethanolamines and glycosylated ceramides, which were poorly detected in MALDI-MSI. In negative ion mode, MALDI favored sulfatides and free fatty acids, whereas NAPA spectra were dominated by signal from phosphatidylethanolamines. The complementarity in lipid coverages between the NAPA- and MALDI-MSI platforms presents the possibility of selective lipid analysis and imaging dependent upon which platform is used. Nanofabrication of the NAPA platform offers better uniformity compared to MALDI, and the wider dynamic range offered by NAPA promises improved quantitation in imaging.

Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer.

A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. Here, we present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 µm for the commercial configuration down to ~9 µm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-µm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed.