Lipid biochemical and structural changes after TBI could help highlighting molecules that can be used to determine the severity of such injuries as well as to evaluate the efficacy of potential treatments.

No abstract

The well-characterized cellular and structural components of the kidney show distinct regional compositions and distribution of lipids. In order to more fully analyze the renal lipidome we developed a matrix-assisted laser desorption/ionization mass spectrometry approach for imaging that may be used to pinpoint sites of changes from normal in pathological conditions. This was accomplished by implanting sagittal cryostat rat kidney sections with a stable, quantifiable and reproducible uniform layer of silver using a magnetron sputtering source to form silver nanoparticles. Thirty-eight lipid species including 7 ceramides, 8 diacylglycerols, 22 triacylglycerols, and cholesterol were detected and imaged in positive ion mode. Thirty-six lipid species consisting of, 7 sphingomyelins, 10 phosphatidylethanolamines, 1 phosphatidylglycerol, 7 phosphatidylinositols and 11 sulfatides, were imaged in negative ion mode for a total of seventy-four high resolution lipidome maps of the normal kidney. Thus, our approach is a powerful tool not only for studying structural changes in animal models of disease, but also for diagnosing and tracking stages of disease in human kidney tissue biopsies.

Mass spectrometry imaging (MSI) of tissue implanted with silver nanoparticulate (AgNP) matrix generates reproducible imaging of lipids in rodent models of disease and injury. Gas-phase production and acceleration of size-selected 8 nm AgNP is followed by controlled ion beam rastering and soft landing implantation of 500 eV AgNP into tissue. Focused 337 nm laser desorption produces high quality images for most lipid classes in rat brain tissue (in positive mode: galactoceramides, diacylglycerols, ceramides, phosphatidylcholines, cholesteryl ester, and cholesterol, and in negative ion mode: phosphatidylethanolamides, sulfatides, phosphatidylinositol, and sphingomyelins). Image reproducibility in serial sections of brain tissue is achieved within <10% tolerance by selecting argentated instead of alkali cationized ions. The imaging of brain tissues spotted with pure standards was used to demonstrate that Ag cationized ceramide and diacylglycerol ions are from intact, endogenous species. In contrast, almost all Ag cationized fatty acid ions are a result of fragmentations of numerous lipid types having the fatty acid as a subunit. Almost no argentated intact fatty acid ions come from the pure fatty acid standard on tissue. Graphical Abstract ᅟ.

Polycyclic aromatic hydrocarbons (PAH) are metabolized to electrophiles that can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAH-treated mouse skin. Depurinating adducts account for 71% of all DNA adducts formed in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls for the presence and quantities of the depurinated BP-adducted DNA bases, 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade). Since these adducted bases originate from reaction of the BP radical cation with double-stranded DNA and not with RNA or denatured DNA, their presence in urine is indicative of DNA damage. Urine samples were fractionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis with laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to coal smoke, but were not detected in urine from the 13 control subjects. Concentrations were estimated to be 60-340 and 0.1-0.6 fmol/mg of creatinine equivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20-300 times the concentration of BP-6-N7Ade in the urine of coal smoke-exposed women, but was not detected in the urine of cigarette smokers. This difference may be due to the remarkably different BP exposures experienced by the two groups of PAH-exposed individuals. These results justify more extensive studies of depurinated BP-adducted DNA bases as potential biomarkers of PAH-associated cancer risk.

Profiling and imaging MALDI mass spectrometry (MS) allows detection and localization of biomolecules in tissue, of which lipids are a major component. However, due to the in situ nature of this technique, complexity of tissue and need for a chemical matrix, the recorded signal is complex and can be difficult to assign. Ion mobility adds a dimension that provides coarse shape information, separating isobaric lipids, peptides, and oligonucleotides along distinct familial trend lines before mass analysis. Previous work using MALDI-ion mobility mass spectrometry to analyze and image lipids has been conducted mainly in positive ion mode, although several lipid classes ionize preferentially in negative ion mode. This work highlights recent data acquired in negative ion mode to detect glycerophosphoethanolamines (PEs), glycerophosphoserines (PSs), glycerophosphoglycerols (PGs), glycerolphosphoinositols (PIs), glycerophosphates (PAs), sulfatides (STs), and gangliosides from standard tissue extracts and directly from mouse brain tissue. In particular, this study focused on changes in ion mobility based upon lipid head groups, composition of radyl chain (# of carbons and double bonds), diacyl versus plasmalogen species, and hydroxylation of species. Finally, a MALDI-ion mobility imaging run was conducted in negative ion mode, resulting in the successful ion mapping of several lipid species.

This paper describes the optimization of sample preparation for MALDI 193-nm photofragment ion time-of-flight mass spectrometry to sequence small to medium-sized peptides from peptide mixtures. We show that matrix additives, such as fructose and phenylbutyric acid have a dramatic effect on the abundance of fragment ions observed in the post-source decay spectra. A dried-droplet MALDI matrix consisting of 1:1 alpha-cyano-4-hydroxycinnamic acid/fructose proves to be an excellent matrix for photodissociation because [M + H]+ ions are formed with low internal energies, and the photofragment ion spectrum contains high abundances of sequence-informative ions. The addition of fructose appears to improve overall sample homogeneity and durability, as compared to conventional alpha-cyano-4-hydroxycinnamic acid dried-droplet preparations. MALDI-TOF photodissociation is then used to selectively sequence the peptides bradykinin (RPPGFSPFR), des-Arg9 bradykinin (RPPGFSPF), and substance P-amide (RPKPQQFFGLM-NH2) from a mixture of five peptides.

Traumatic brain injury (TBI) is a serious public health problem and the leading cause of death in children and young adults. It also contributes to a substantial number of cases of permanent disability. As lipids make up over 50% of the brain mass and play a key role in both membrane structure and cell signaling, their profile is of particular interest. In this study, we show that advanced mass spectrometry imaging (MSI) has sufficient technical accuracy and reproducibility to demonstrate the anatomical distribution of 50 μm diameter microdomains that show changes in brain ceramide levels in a rat model of controlled cortical impact (CCI) 3 days post injury with and without treatment. Adult male Sprague-Dawley rats received one strike and were euthanized 3 days post trauma. Brain MS images showed increase in ceramides in CCI animals compared to control as well as significant reduction in ceramides in CCI treated animals, demonstrating therapeutic effect of a peptide agonist. The data also suggests the presence of diffuse changes outside of the injured area. These results shed light on the extent of biochemical and structural changes in the brain after traumatic brain injury and could help to evaluate the efficacy of treatments.