Significant advances in mass spectrometry imaging (MSI) have pushed the boundaries in obtaining spatial information and quantification in biological samples. Quantitative MSI (qMSI) has typically been challenging to achieve because of matrix and tissue heterogeneity, inefficient analyte extraction, and ion suppression effects, but recent studies have demonstrated approaches to obtain highly robust methods and reproducible results. In this perspective, we share our insights into sample preparation, how the choice of matrix influences sensitivity, construction of calibration curves, signal normalization, and visualization of MSI data. We hope that by articulating these guidelines that qMSI can be routinely conducted while retaining the analytical merits of other mass spectrometry modalities.

Poly(glutamic acid) at low pH self-assembles after incubation at higher temperature into fibrils composed of antiparallel sheets that are stacked in a β2-type structure whose amide carbonyls have bifurcated H-bonds involving the side chains from the next sheet. Oligomers of Glu can also form such structures, and isotope labeling has provided insight into their out-of-register antiparallel structure [ Biomacromolecules 2013 , 14 , 3880 - 3891 ]. In this paper we report IR and VCD spectra and transmission electron micrograph (TEM) images for a series of alternately sequenced oligomers, Lys-(Aaa-Glu)5-Lys-NH2, where Aaa was varied over a variety of polar, aliphatic, or aromatic residues. Their spectral and TEM data show that these oligopeptides self-assemble into different structures, both local and morphological, that are dependent on both the nature of the Aaa side chains and growth conditions employed. Such alternate peptides substituted with small or polar residues, Ala and Thr, do not yield fibrils; but with β-branched aliphatic residues, Val and Ile, that could potentially pack with Glu side chains, these oligopeptides do show evidence of β2-stacking. By contrast, for Leu, with longer side chains, only β1-stacking is seen while with even larger Phe side chains, either β-form can be detected separately, depending on preparation conditions. These structures are dependent on high temperature incubation after reducing the pH and in some cases after sonication of initial fibril forms and reincubation. Some of these fibrillar peptides, but not all, show enhanced VCD, which can offer evidence for formation of long, multistrand, often twisted structures. Substitution of Glu with residues having selected side chains yields a variety of morphologies, leading to both β1- and β2-structures, that overall suggests two different packing modes for the hydrophobic side chains depending on size and type.

This article is available online at http://www.jlr.org be viewed relative to their location and intensity. As recently reviewed (9), MALDI-MSI of lipids has been quite successful, given that lipids are abundant in biological tissues. Additionally, with the exception of cardiolipins and gangliosides, lipids have molecular masses between 200 and 1,000 Da, an optimal operating mass range of MALDI-TOF instruments to achieve high mass resolving power and maintain accurate mass measurements.Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, lysosomal storage disorder caused by mutations of the encoding regions of genes either for the lysosomal transmembrane protein NPC1 or the cholesterol-binding glycoprotein NPC2 (10), two proteins that work in tandem to mobilize cholesterol through the late endosomal/lysosomal system (11)(12)(13)(14). As a result of the primary genetic defect, unesterified cholesterol and glycosphingolipids accumulate in late endosomes and lysosomes. Specifically, dysfunction of these proteins causes the inability to metabolize and traffic contents, and eventually cellular death. Cerebellar Purkinje neurons appear to be most susceptible though the cerebral cortex; thalamus and hippocampus also demonstrate neuronal loss (15)(16)(17)(18)(19). Systemic downstream effects include oxidative stress (20), defective calcium signaling (21), and neuroinflammation (22). The clinical phenotype of NPC is broad and includes vertical supranuclear gaze palsy, tremors, ataxia, and early dementia, and ultimately is fatal (23). While several studies have looked at transcript and protein-level alterations in NPC1 (22,(24)(25)(26)(27), less is known about alterations in the lipid landscape in the disease. Early work on lipid accumulation demonstrated that several tissues are affected (28-30). A targeted MS-based profiling study was reported in which different sphingolipids species were observed to be differential in the null NPC1 mouse model (31) as well as in the recently generated I1061T point mutant model (32).Abstract Mass spectrometry imaging (MSI) is a tool to rapidly map the spatial location of analytes without the need for tagging or a reporter system. Niemann-Pick disease type C1 (NPC1) is a neurodegenerative, lysosomal storage disorder characterized by accumulation of unesterified cholesterol and sphingolipids in the endo-lysosomal system. Here, we use MSI to visualize lipids including cholesterol in cerebellar brain tissue from the NPC1 symptomatic mouse model and unaffected controls. To complement the imaging studies, a data-processing pipeline was developed to generate consensus mass spectra, thereby using both technical and biological image replicates to assess differences. The consensus spectra are used to determine true differences in lipid relative abundance; lipid distributions can be determined in an unbiased fashion without prior knowledge of location. We show the cerebellar distribution of gangliosides GM1, GM2, and GM3, including variants of lipid chain length. We also performed MALDI-...

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Altered lipid metabolism is one of the hallmarks of cancer. Cellular proliferation and de novo synthesis of lipids are related to cancer progression. In this study, we evaluated the lipidomic profile of two-dimensional (2D) monolayer and multicellular tumor spheroids from the HCT 116 colon carcinoma cell line. We utilized serial trypsinization on the spheroid samples to generate three cellular populations representing the proliferative, quiescent, and necrotic regions of the spheroid. This analysis enabled a comprehensive identification and quantification of lipids produced in each of the spheroid layer and 2D cultures. We show that lipid subclasses associated with lipid droplets form in oxygen-restricted and acidic regions of spheroids and are produced at higher levels than in 2D cultures. Additionally, sphingolipid production, which is implicated in cell death and survival pathways, is higher in spheroids relative to 2D cells. Finally, we show that increased numbers of lipids comprised of polyunsaturated fatty acids (PUFAs) are produced in the quiescent and necrotic regions of the spheroid. The lipidomic signature for each region and cell culture type highlights the importance of understanding the spatial aspects of cancer biology. These results provide additional lipid biomarkers in the tumor microenvironment that can be further studied to target pivotal lipid production pathways.