Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolutions and 20 pixels/s throughout molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 ×10 μm at the sample surface. General image performance was assessed using murine kidney and brain *

Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomolecules in tissue. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables approximately a ~270% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn-backbone isomers, acyl chain isomers, as well as double bond positional and geometric isomers are demonstrated. As a proof-of-concept, in situ separation and imaging of lipid isomers with distinct spatial distributions was performed using tissue sections from a whole-body mouse pup. File list (2) download file view on ChemRxiv 200813_Manuscript_v2_FINAL.pdf (756.02 KiB) download file view on ChemRxiv 200813_Supplemental_v2_FINAL.pdf (1.08 MiB)

Background Trimethylamine‐N‐oxide ( TMAO ), a diet‐derived, gut microbial–host cometabolite, has been associated with adverse cardiovascular outcomes in patient populations; however, evidence is lacking from prospective studies conducted in general populations and non‐Western populations. Methods and Results We evaluated urinary levels of TMAO and its precursor metabolites (ie, choline, betaine, and carnitine) in relation to risk of coronary heart disease ( CHD ) among Chinese adults in a nested case–control study, including 275 participants with incident CHD and 275 individually matched controls. We found that urinary TMAO , but not its precursors, was associated with risk of CHD . The odds ratio for the highest versus lowest quartiles of TMAO was 1.91 (95% CI, 1.08–3.35; P trend =0.008) after adjusting for CHD risk factors including obesity, diet, lifestyle, and metabolic diseases and 1.75 (95% CI, 0.96–3.18; P trend =0.03) after further adjusting for potential confounders or mediators including central obesity, dyslipidemia, inflammation, and intake of seafood and deep‐fried meat or fish, which were associated with TMAO level in this study. The odds ratio per standard deviation increase in log‐ TMAO was 1.30 (95% CI, 1.03–1.63) in the fully adjusted model. A history of diabetes mellitus modified the TMAO – CHD association. A high TMAO level (greater than or equal to versus lower than the median) was associated with odds ratios of 6.21 (95% CI, 1.64–23.6) and 1.56 (95% CI, 1.00–2.43), respectively, among diabetic and nondiabetic participants ( P interaction =0.02). Diabetes mellitus status also modified the associations of choline, betaine, and carnitine with risk of CHD ; significant positive associations were found among diabetic participants, but null associations were noted among total and nondiabetic participants. Conclusions Our study suggests that TMAO may accelerate the development of CHD , highlighting the importance of diet–gut microbiota–host interplay in cardiometabolic health.

SUMMARY Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia and a critical threat due to multidrug resistance. The A. baumannii outer membrane is an asymmetric lipid bilayer composed of inner leaflet glycerophospholipids and outer leaflet lipooligosaccharides. Deleting mlaF of the maintenance of lipid asymmetry (Mla) system causes A. baumannii to become more susceptible to pulmonary surfactants and antibiotics and decreases bacterial survival in the lungs of mice. Spontaneous suppressor mutants isolated from infected mice contain an IS Aba11 insertion upstream of the ispB initiation codon, an essential isoprenoid biosynthesis gene. The insertion restores antimicrobial resistance and virulence to Δ mlaF . The suppressor strain increases lipooligosaccharides, suggesting that the mechanism involves balancing the glycerophospholipids/lipooligosaccharides ratio on the bacterial surface. An identical insertion exists in an extensively drug-resistant A. baumannii isolate, demonstrating its clinical relevance. These data show that the stresses bacteria encounter during infection select for genomic rearrangements that increase resistance to antimicrobials.

Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion mobility spectrometry imaging platform. This prototype MALDI timsTOF instrument is capable of 10 µm spatial resolutions and 20 pixels/s throughput molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 x 10 µm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high spatial resolution imaging data can be generated rapidly with mass measurement errors < 5 ppm and ~40,000 resolving power. Initial TIMS-based imaging experiments were performed on whole body mouse pup tissue demonstrating the separation of closely isobaric [PC(32:0)+Na]⁺and [PC(34:3)+H]⁺(3 mDa mass difference) in the gas-phase. We have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixtures of lipid adducts. The combination of high spatial resolution and throughput imaging capabilities with high-performance TIMS separations provides a uniquely tunable platform to address many challenges associated with advanced molecular imaging applications.