2,2'-{(1E,1'E)-[Ethane-1,2-diylbis(aza-nylyl-idene)]bis-(methanylyl-idene)}bis-(4-iodo-phenol).

The title compound, C20H14I2N2O2, a di-iodo-Schiff base, crystallizes in space group Pbca with one mol-ecule per asymmetric unit. The mol-ecular structure reveals two intra-molecular O-H⋯N hydrogen bonds that give the mol-ecule a twisted structure with non-coplanar rings. In the crystal structure, the mol-ecular packing is stabilized by π-π stacking, hydrogen- and halogen-bonding (C-H⋯I; O⋯I) inter-actions.

Mass Spectrometry-Based Analysis of Lipid Involvement in Alzheimer's Disease Pathology-A Review.

Irregularities in lipid metabolism have been linked to numerous neurodegenerative diseases. The roles of abnormal brain, plasma, and cerebrospinal fluid (CSF) lipid levels in Alzheimer's disease (AD) onset and progression specifically have been described to a great extent in the literature. Apparent hallmarks of AD include, but are not limited to, genetic predisposition involving the APOE Ɛ4 allele, oxidative stress, and inflammation. A common culprit tied to many of these hallmarks is disruption in brain lipid homeostasis. Therefore, it is important to understand the roles of lipids, under normal and abnormal conditions, in each process. Lipid influences in processes such as inflammation and blood-brain barrier (BBB) disturbance have been primarily studied via biochemical-based methods. There is a need, however, for studies focused on uncovering the relationship between lipid irregularities and AD by molecular-based quantitative analysis in transgenic animal models and human samples alike. In this review, mass spectrometry as it has been used as an analytical tool to address the convoluted relationships mentioned above is discussed. Additionally, molecular-based mass spectrometry strategies that should be used going forward to further relate structure and function relationships of lipid irregularities and hallmark AD pathology are outlined.

Ethanol-Fixed, Paraffin-Embedded Tissue Imaging: Implications for Alzheimer's Disease Research.

Mass spectrometry imaging (MSI) is rapidly becoming a crucial tool in disease research. Fresh-frozen tissue is ideal for MSI because the protein and lipid structures are undisturbed by chemical fixatives; however, that means long-term preservation is limited. Formalin-fixed paraffin-embedded tissue has a virtually infinite shelf life, but whole proteins are difficult or impossible to image directly. To bridge this gap, we examine the use of ethanol-fixed, paraffin-embedded (EFPE) tissue for the localization of intact proteins and lipids and comment on implications in Alzheimer's disease (AD) research. The new sample preparation methods for EFPE tissues have allowed us to greatly broaden the information we can extract from MSI experiments. Our methods involve a xylene-free deparaffination for lipid analysis and an intact protein method for visualizing amyloid-beta plaques from human AD brain tissue. This unique combination streamlines the MSI sample preparation process while allowing for the most biologically and pathologically relevant information to be extracted from a single tissue source.

Rapid method towards proteomic analysis of dried blood spots by MALDI mass spectrometry.

Neonatal dried blood spots (DBS) are routinely utilized in the clinical setting as a diagnostic tool for various genetic disorders and infectious diseases. DBS allow for minimally invasive, small volume blood collection and are stored at room temperature. Neonatal whole blood and serum samples can be important in determining genetic risk factors and predicting infantile disease; however, at the present time, limited methods exist for rapidly analyzing DBS samples for their proteomic profile, years after samples have been collected. A novel method is presented for the extraction and analysis of target proteins and peptides from neonatal DBS using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Extraction parameters were optimized to achieve ideal signal intensity and resolution to obtain protein identifications. Samples were extracted from filter paper with 0.1% TFA in H2O for 72 h. The extract was subjected to enzymatic digestion, spotted on an ITO-coated glass slide, and washed in order to remove salts. Analysis of extracted blood spots from ten newborns was completed. Similarities and differences in the proteomic profile of the washed extracts are presented, herein, to verify the viability of this method for analysis of dated DBS samples. This method allows for analysis of DBS samples years after collection and can be utilized to correlate diseases or disorders manifesting later in life with potential risk factors presenting in the proteomic profile of the DBS collected at time of birth.

Characterization of Proteins Present in Isolated Senile Plaques from Alzheimer's Diseased Brains by MALDI-TOF MS with MS/MS.

The increase of insoluble senile plaques in the brain is a primary hallmark of Alzheimer's disease. The usefulness of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with tandem MS for the characterization of senile plaques from AD brains and the relevance of the components identified to furthering AD research using MS is discussed. Thirty-three components were reproducibly observed within tryptic aliquots of senile plaques from two different AD brains after sample preparation optimization. Additionally, this is one of the first accounts of LIFT being utilized for the direct sequencing of peptides from isolated senile plaques. While many of the species observed coisolated within senile plaques have been linked to AD etiology, if only speculatively, this is the first instance that many of them have been demonstrated to be a part of the plaques themselves. This work is the first step in determining the potential roles that the species may have in the aggregation or proliferation of the plaques.

Incubation with Cu(II) and Zn(II) salts enhances MALDI-TOF mass spectra of amyloid-beta and α-synuclein toward in vivo analysis.

Insoluble senile plaque aggregates are indicative of Alzheimer's disease pathology. A similar phenomenon occurs in Parkinson's disease with the build-up of Lewy bodies. The analysis of senile plaques, and other brain samples, from Alzheimer's disease and Parkinson's disease patients by matrix-assisted laser desorption/ionization mass spectrometry has advantages but also presents obstacles because of the nature of the processes utilized in isolation procedures and storage. Salts, buffers, and detergents necessary in the isolation of biological species may cause adducts and ion suppression that convolute the spectra obtained. We previously determined that amyloid-beta from isolated senile plaque deposits fragment similarly to the synthetic 40 and 42 amino acid peptide when analyzed by matrix-assisted laser desorption/ionization mass spectrometry. In addition, α-synuclein also fragments predictably by in-source decay. This provides information that may be applied to the identification and localization of amyloid-beta and α-synuclein in senile plaques and intact tissue sections. Ion suppression must still be accounted for when analyzing biological samples, which makes identifying fragments at lower abundance difficult. The addition of certain transition-metal salts (Cu(II), Zn(II)) to the sample prior to analysis serves to "clean" the spectra and allow the peptide fragments produced to be observed with a much higher signal to noise and occasionally, improved resolution. We present a systematic study of incubation with different metal salts and their impact on the quality of the spectra, as well as the role of the binding of the metals to the model biological compounds, obtained for synthetic amyloid-beta, synthetic α-synuclein, and isolated senile plaques. The optimized sample preparation methods presented will provide for simpler and more thorough identification of these biologically relevant species in human-derived samples.

Molecular Mapping Alzheimer's Disease: MALDI Imaging of Formalin-fixed, Paraffin-embedded Human Hippocampal Tissue.

A method for the molecular mapping of formalin-fixed, paraffin-embedded human hippocampal tissue affected by Alzheimer's disease (AD) is presented. This approach utilizes imaging mass spectrometry (IMS) with matrix-assisted laser desorption/ionization (MALDI). The usefulness of this technique in comparing diseased versus nor mal tissue at the molecular level while continuing to maintain topological and morphological integrity is evident in the preliminary findings. The critical correlation of the deparaffination, washing, matrix deposition, and analysis steps in handling the tissue sections and how these steps impact the successful mapping of human hippocampal tissue is clearly demonstrated. By use of this technique we have been able to identify several differences between the hippocampal AD tissue and the control hippocampal tissue. From the observed peptide clip masses we present preliminary identifications of the amyloid-beta peptides known to be prominent in the brains of those with AD. We have obtained high-resolution mass spectra and mass images with 100µm spatial resolution. Future experiments will couple this work with MALDI LIFT experiments to enable top down proteomics of fresh frozen tissue, which is not possible with paraffin-embedded tissues.

Laser-Induced In-Source Decay Applied to the Determination of Amyloid-Beta in Alzheimer's Brains.

A method for the analysis of amyloid-beta peptides in isolated plaques and intact tissue sections affected by Alzheimer's disease (AD) is presented. This method employs matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry and the inherent laser-induced in-source decay (ISD) that occurs coupled with imaging mass spectrometry (IMS) to investigate the composition of these samples eliminating the need for other confirmational MS/MS techniques. These results demonstrate this technique's usefulness for the identification of amyloid-beta peptides in tissue and isolated senile plaques from AD patients using the reproducible fragmentation pattern demonstrated via the laser-induced ISD of synthetic amyloid-beta peptide clips (1-40, 1-42). Clear differences between the hippocampal AD tissue and the control hippocampal tissue regarding the presence of amyloid-beta have been identified. These are based on laser-induced ISD of standard amyloid-beta clips as controls as well as the analysis of isolated senile plaques as a confirmation before tissue analysis. Using the resulting observed peptide clip masses from the control data, we present mass spectrometry based identification of the amyloid-beta peptides in both isolated plaques and hippocampal regions of those patients diagnosed with AD.