No abstract

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.

The utility of post-source decay (PSD) matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS) was investigated for the structural analysis of phosphatidylcholine (PC). PC did not produce detectable negative molecular ion from MALDI, but positive ions were observed as both [PCϩH] ϩ and [PCϩNa] ϩ . The PSD spectra of the protonated PC species contained only one fragment corresponding to the head group (m/z 184), while the sodiated precursors produced many fragment ions, including those derived from the loss of fatty acids. The loss of fatty acid from the C-1 position (sn-1) of the glycerol backbone was favored over the loss of fatty acid from the C-2 position (sn-2). Ions emanating from the fragmentation of the head group (phosphocholine) included ϩ , ϩ and ϩ , which corresponded to the loss of trimethylamine (TMA), non-sodiated choline phosphate and sodiated choline phosphate, respectively. Other fragments reflecting the structure of the head group were observed at m/z 183, 146 and 86. The difference in the fragmentation patterns for the PSD of [PCϩNa] ϩ compared to [PCϩH] ϩ is attributed to difference in the binding of Na ϩ and H ϩ . While the proton binds to a negatively charged oxygen of the phosphate group, the sodium ion can be associated with several regions of the PC molecule. Hence, in the sodiated PC, intermolecular interaction of the negatively charged oxygen of the phosphate group, along with sodium association at multiple sites, can lead to a complex and characteristic ion fragmentation pattern. The preferential loss of sn-1 fatty acid group could be explained by the formation of an energetically favorable six-member A s major constituents of cell membranes, phospholipids (PL) have an essential role in regulating biophysical properties, protein sorting and cell signaling pathways. Structurally, PLs are diacyl(alkyl)glycerols esterified to a phosphate-containing polar head group. The nature of the head group dictates the particular PL class. PL molecular species are defined by the nature of the acyl (alkyl) residues attached to the C-1 (also called sn-1, using stereospecific numbering) and C-2 positions (sn-2) of the glycerol backbone. The molecular species are uniquely and differentially distributed among different tissues [1,2], and because of their importance in regulating development, function and adaptation [3][4][5], analysis of PL molecular species and their alterations is of considerable interest in many areas of biological research.Thin layer chromatography (TLC) [6 -8] and normal-phase high performance liquid chromatography (HPLC) [9,10] are well suited for the separation of PL classes, whereas the separation of molecular species within each class may be accomplished using gas chromatography (GC) [11,12] or reversed-phase HPLC [13][14][15] (see [5] for a review on lipid molecular species analysis). Conversion of PL species to GCsuitable derivatives is laborious and time consuming, and often results in analyte loss. Moreover, HPLC can

Objective: Stroke is the main cause of adult disability in the world, leaving more than half of the patients dependent on daily assistance. Understanding the post-stroke biochemical and molecular changes are critical for patient survival and stroke management. The aim of this work was to investigate the photo-thrombotic ischemic stroke in male rats with particular focus on biochemical and elemental changes in the primary stroke lesion in the somatosensory cortex and surrounding areas, including the corpus callosum.Materials and Methods: FT-IR imaging spectroscopy and LA-ICPMS techniques examined stroke brain samples, which were compared with standard immunohistochemistry studies.Results: The FTIR results revealed that in the lesioned gray matter the relative distribution of lipid, lipid acyl and protein contents decreased significantly. Also at this locus, there was a significant increase in aggregated protein as detected by high-levels Aβ1-42. Areas close to the stroke focus experienced decrease in the lipid and lipid acyl contents associated with an increase in lipid ester, olefin, and methyl bio-contents with a novel finding of Aβ1-42 in the PL-GM and L-WM. Elemental analyses realized major changes in the different brain structures that may underscore functionality.Conclusion: In conclusion, FTIR bio-spectroscopy is a non-destructive, rapid, and a refined technique to characterize oxidative stress markers associated with lipid degradation and protein denaturation not characterized by routine approaches. This technique may expedite research into stroke and offer new approaches for neurodegenerative disorders. The results suggest that a good therapeutic strategy should include a mechanism that provides protective effect from brain swelling (edema) and neurotoxicity by scavenging the lipid peroxidation end products.

We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products. In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution. Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components. To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay. This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis.