Jeffrey M. Spraggins

MALDI imaging of lipid biochemistry in tissues by mass spectrometry.

As a result of recent advances, remarkable images revealing the distribution of complex lipids in tissues are now generated by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Lipids are amphipathic biomolecules with hydrophobic structural characteristics made by either an initial anion thioester condensation reaction (fatty acid synthase) or by carbocation condensation of branched chain pyrophosphate intermediates (isoprene pathway).1 Lipids play essential roles in cellular function including the self-assembly of phospholipids to form the constitutive outer and inner membrane bilayer of every living cell. Specific components of these membrane phospholipids include species that contain esterified arachidonate that can be enzymatically released to a free acid and transformed to potent signaling molecules (prostaglandins, leukotrienes) with myriad biological effects. The lipid cholesterol is an essential component of bilayer membranes that has a complicated, yet highly regulated biosynthesis. Elevation of cholesterol levels (predominantly as cholesteryl esters) in blood has been implicated in heart disease and is commonly monitored in consideration of human health. Some lipid molecules play the central role in biochemical energy storage in the form of triacylglycerol molecules stored in lipid bodies within most all cells. Mass spectrometry has historically been a tool of choice in biochemical studies of lipids. The sensitivity and specificity of mass spectral data are useful to sort out the complexity of lipid structures to begin to follow biological changes. While techniques such as fluorescence confocal microscopy or ability to engineer proteins that can be expressed in cells with fluorescent tags have become the mainstream of modern biochemical research, such techniques are not amenable to most lipids due to the relatively small size of the lipid molecules and the dynamic nature of their structure in the cell. Recent developments in MALDI IMS have merged specificity of lipid identification with two-dimensional molecular mapping to enable biochemical studies of lipids across regions of a biological tissue. Several significant reasons for the success of MALDI IMS applied to lipid imaging have emerged. The first is the high abundance of various lipids in biological tissues because these hydrophobic molecules constitute the external and internal defining membranes of each cell. These membranes are almost exclusively bilayers composed of phospholipids, sphingolipids, and cholesterol that are closely packed in high local concentrations to render the membrane only semipermeable to water. A second reason is that many lipids, e.g. phospholipids, are already ionized as either phosphate anions or nitrogen centered cations and generate abundant positive or negative ions during the MALDI process. An equally important factor in the success of MALDI IMS of lipids is that the molecular weight of these biomolecules is generally below 1,000 Da, which is an optimal mass range for the most sensitive operation of modern mass spectrometers. Additionally this low molecular weight facilitates diffusion of lipids into a matrix crystal driven by the high concentration of the lipid within the microstructure of the tissue. Because of these fundamental factors coupled with the exciting potential of MALDI IMS, lipid molecules have been frequently used as substrates for the advancement of IMS methodology and instrumentation. Research groups that utilize secondary ion mass spectrometry (SIMS) imaging have embraced lipid biochemistry by moving from inorganic to biological applications, development of larger particle size beams and demonstrations of sub-micron lateral resolution.2,3 Similar development and implementation of instrumentation for MALDI IMS has leveraged lipid diversity, abundance and contrast in rodent brain samples to achieve advancements in technology.4-8 The development of different matrices useful for MALDI IMS,9-16 different methods of matrix application17-23 and different matrix modifiers24,25 have been employed in MALDI IMS experiments to establish the value and parameters of these method modifications for lipid analysis. Advances in biology have been a direct result of our ability to observe biochemical events at the micron and submicron regimes within a tissue. Having a sensitive technique that reveals molecular structure information about specific lipids in a tissue with 10-50 μm resolution and provides information relative to concentration of that lipid, has already provided insight into lipid biochemistry at the tissue level. Since lipids are products of complex, intertwined enzymatic processes, MALDI IMS data reveals the integrated solution to complex reaction pathways that define the living cell in terms of lipid biochemistry. It has become apparent to a host of scientists converging into the use of MALDI IMS from fields as diverse as neuroscience, chemistry, and instrument development that there is a richness and complexity of lipid biochemistry suggested by the exquisite, molecule specific MALDI images created in the course of developing this technology. Many reviews have focused on the technological developments of MALDI IMS of lipids with respect to the issues mentioned above.2,26,27 This review focuses on the lipid biochemistry revealed by MALDI IMS.

Enhanced Sensitivity for High Spatial Resolution Lipid Analysis by Negative Ion Mode Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry

We have achieved enhanced lipid imaging to a ~10 μm spatial resolution using negative ion mode matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry, sublimation of 2,5-dihydroxybenzoic acid as the MALDI matrix, and a sample preparation protocol that uses aqueous washes. We report on the effect of treating tissue sections by washing with volatile buffers at different pHs prior to negative ion mode lipid imaging. The results show that washing with ammonium formate, pH 6.4, or ammonium acetate, pH 6.7, significantly increases signal intensity and number of analytes recorded from adult mouse brain tissue sections. Major lipid species measured were glycerophosphoinositols, glycerophosphates, glycerolphosphoglycerols, glycerophosphoethanolamines, glycerophospho-serines, sulfatides, and gangliosides. Ion images from adult mouse brain sections that compare washed and unwashed sections are presented and show up to 5-fold increases in ion intensity for washed tissue. The sample preparation protocol has been found to be applicable across numerous organ types and significantly expands the number of lipid species detectable by imaging mass spectrometry at high spatial resolution.

MALDI FTICR IMS of Intact Proteins: Using Mass Accuracy to Link Protein Images with Proteomics Data

AbstractMALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here, we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (~75,000 at m/z 5000) and accuracy (<5ppm) for proteins up to ~12kDa, enabling identification based on correlation with LC-MS/MS proteomics data. Analysis of rat brain tissue was performed as a proof-of-concept highlighting the capabilities of this approach by imaging and identifying a number of proteins including N-terminally acetylated thymosin β4 (m/z 4,963.502, 0.6ppm) and ATP synthase subunit ε (m/z 5,636.074, –2.3ppm). MALDI FTICR IMS was also used to differentiate a series of oxidation products of S100A8 (m/z 10,164.03, –2.1ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 – M37O/C42O3 (m/z 10228.00, –2.6ppm) was found to co-localize with bacterial microcolonies at the center of infectious foci. The ability of MALDI FTICR IMS to distinguish S100A8 modifications is critical to understanding calprotectin’s roll in nutritional immunity. Graphical Abstractᅟ

Diabetic nephropathy induces alterations in the glomerular and tubule lipid profiles.

Diabetic nephropathy (DN) is a major life-threatening complication of diabetes. Renal lesions affect glomeruli and tubules, but the pathogenesis is not completely understood. Phospholipids and glycolipids are molecules that carry out multiple cell functions in health and disease, and their role in DN pathogenesis is unknown. We employed high spatial resolution MALDI imaging MS to determine lipid changes in kidneys of eNOS−/− db/db mice, a robust model of DN. Phospholipid and glycolipid structures, localization patterns, and relative tissue levels were determined in individual renal glomeruli and tubules without disturbing tissue morphology. A significant increase in the levels of specific glomerular and tubular lipid species from four different classes, i.e., gangliosides, sulfoglycosphingolipids, lysophospholipids, and phosphatidylethanolamines, was detected in diabetic kidneys compared with nondiabetic controls. Inhibition of nonenzymatic oxidative and glycoxidative pathways attenuated the increase in lipid levels and ameliorated renal pathology, even though blood glucose levels remained unchanged. Our data demonstrate that the levels of specific phospho- and glycolipids in glomeruli and/or tubules are associated with diabetic renal pathology. We suggest that hyperglycemia-induced DN pathogenic mechanisms require intermediate oxidative steps that involve specific phospholipid and glycolipid species.

Non-small cell lung cancer is characterized by dramatic changes in phospholipid profiles

Non‐small cell lung cancer (NSCLC) is the leading cause of cancer death globally. To develop better diagnostics and more effective treatments, research in the past decades has focused on identification of molecular changes in the genome, transcriptome, proteome, and more recently also the metabolome. Phospholipids, which nevertheless play a central role in cell functioning, remain poorly explored. Here, using a mass spectrometry (MS)‐based phospholipidomics approach, we profiled 179 phospholipid species in malignant and matched non‐malignant lung tissue of 162 NSCLC patients (73 in a discovery cohort and 89 in a validation cohort). We identified 91 phospholipid species that were differentially expressed in cancer versus non‐malignant tissues. Most prominent changes included a decrease in sphingomyelins (SMs) and an increase in specific phosphatidylinositols (PIs). Also a decrease in multiple phosphatidylserines (PSs) was observed, along with an increase in several phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species, particularly those with 40 or 42 carbon atoms in both fatty acyl chains together. 2D‐imaging MS of the most differentially expressed phospholipids confirmed their differential abundance in cancer cells. We identified lipid markers that can discriminate tumor versus normal tissue and different NSCLC subtypes with an AUC (area under the ROC curve) of 0.999 and 0.885, respectively. In conclusion, using both shotgun and 2D‐imaging lipidomics analysis, we uncovered a hitherto unrecognized alteration in phospholipid profiles in NSCLC. These changes may have important biological implications and may have significant potential for biomarker development.

Next-generation technologies for spatial proteomics: Integrating ultra-high speed MALDI-TOF and high mass resolution MALDI FTICR imaging mass spectrometry for protein analysis.

MALDI imaging mass spectrometry is a powerful analytical tool enabling the visualization of biomolecules in tissue. However, there are unique challenges associated with protein imaging experiments including the need for higher spatial resolution capabilities, improved image acquisition rates, and better molecular specificity. Here we demonstrate the capabilities of ultra‐high speed MALDI‐TOF and high mass resolution MALDI FTICR IMS platforms as they relate to these challenges. High spatial resolution MALDI‐TOF protein images of rat brain tissue and cystic fibrosis lung tissue were acquired at image acquisition rates >25 pixels/s. Structures as small as 50 μm were spatially resolved and proteins associated with host immune response were observed in cystic fibrosis lung tissue. Ultra‐high speed MALDI‐TOF enables unique applications including megapixel molecular imaging as demonstrated for lipid analysis of cystic fibrosis lung tissue. Additionally, imaging experiments using MALDI FTICR IMS were shown to produce data with high mass accuracy (<5 ppm) and resolving power (∼75 000 at m/z 5000) for proteins up to ∼20 kDa. Analysis of clear cell renal cell carcinoma using MALDI FTICR IMS identified specific proteins localized to healthy tissue regions, within the tumor, and also in areas of increased vascularization around the tumor.

A Derivatization and Validation Strategy for Determining the Spatial Localization of Endogenous Amine Metabolites in Tissues using MALDI Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) studies increasingly focus on endogenous small molecular weight metabolites and consequently bring special analytical challenges. Since analytical tissue blanks do not exist for endogenous metabolites, careful consideration must be given to confirm molecular identity. Here, we present approaches for the improvement in detection of endogenous amine metabolites such as amino acids and neurotransmitters in tissues through chemical derivatization and matrix-assisted laser desorption/ionization (MALDI) IMS. Chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) was used to improve sensitivity and specificity. CA was applied to the tissue via MALDI sample targets precoated with a mixture of derivatization reagent and ferulic acid as a MALDI matrix. Spatial distributions of chemically derivatized endogenous metabolites in tissue were determined by high-mass resolution and MS(n) IMS. We highlight an analytical strategy for metabolite validation whereby tissue extracts are analyzed by high-performance liquid chromatography (HPLC)-MS/MS to unambiguously identify metabolites and distinguish them from isobaric compounds.

Phospholipid profiling identifies acyl chain elongation as a ubiquitous trait and potential target for the treatment of lung squamous cell carcinoma

Lung cancer is the leading cause of cancer death. Beyond first line treatment, few therapeutic options are available, particularly for squamous cell carcinoma (SCC). Here, we have explored the phospholipidomes of 30 human SCCs and found that they almost invariably (in 96.7% of cases) contain phospholipids with longer acyl chains compared to matched normal tissues. This trait was confirmed using in situ 2D-imaging MS on tissue sections and by phospholipidomics of tumor and normal lung tissue of the L-IkkαKA/KA mouse model of lung SCC. In both human and mouse, the increase in acyl chain length in cancer tissue was accompanied by significant changes in the expression of acyl chain elongases (ELOVLs). Functional screening of differentially expressed ELOVLs by selective gene knockdown in SCC cell lines followed by phospholipidomics revealed ELOVL6 as the main elongation enzyme responsible for acyl chain elongation in cancer cells. Interestingly, inhibition of ELOVL6 drastically reduced colony formation of multiple SCC cell lines in vitro and significantly attenuated their growth as xenografts in vivo in mouse models. These findings identify acyl chain elongation as one of the most common traits of lung SCC discovered so far and pinpoint ELOVL6 as a novel potential target for cancer intervention.

Targeted Multiplex Imaging Mass Spectrometry with Single Chain Fragment Variable (scfv) Recombinant Antibodies

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

MALDI Imaging Mass Spectrometry Spatially Maps Age-Related Deamidation and Truncation of Human Lens Aquaporin-0.

PURPOSE To spatially map human lens Aquaporin-0 (AQP0) protein modifications, including lipidation, truncation, and deamidation, from birth through middle age using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). METHODS Human lens sections were water-washed to facilitate detection of membrane protein AQP0. We acquired MALDI images from eight human lenses ranging in age from 2 months to 63 years. In situ tryptic digestion was used to generate peptides of AQP0 and peptide images were acquired on a 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Peptide extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searched to identify peptides observed in MALDI imaging experiments. RESULTS Unmodified, truncated, and fatty acid-acylated forms of AQP0 were detected in protein imaging experiments. Full-length AQP0 was fatty acid acylated in the core and cortex of young (2- and 4-month) lenses. Acylated and unmodified AQP0 were C-terminally truncated in older lens cores. Deamidated tryptic peptides (+0.9847 Da) were mass resolved from unmodified peptides by FTICR MS. Peptide images revealed differential localization of un-, singly-, and doubly-deamidated AQP0 C-terminal peptide (239-263). Deamidation was present at 4 months and increases with age. Liquid chromatography-MS/MS results indicated N246 undergoes deamidation more rapidly than N259. CONCLUSIONS Results indicated AQP0 fatty acid acylation and deamidation occur during early development. Progressive age-related AQP0 processing, including deamidation and truncation, was mapped in human lenses as a function of age. The localization of these modified AQP0 forms suggests where AQP0 functions may change throughout lens development and aging.