Egoitz Astigarraga

Profiling and Imaging of Lipids on Brain and Liver Tissue by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Using 2-Mercaptobenzothiazole as a Matrix

2-Mercaptobenzothiazole (MBT) is employed for the first time as a matrix for the analysis of lipids from tissue extracts using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We demonstrate that the performance of MBT is superior to that of the matrixes commonly employed for lipids, due to its low vapor pressure, its low acidity, and the formation of small crystals, although because of the strong background at low m/z, it precludes detection of species below approximately 500 Da. This inconvenience can be partly overcome with the formation of Cs adducts. Using a polymer-based dual calibration, a mass accuracy of approximately 10 ppm in lipid extracts and of approximately 80 ppm in tissues is achieved. We present spectra from liver and brain lipid extracts where a large amount of lipid species is identified, in both positive and negative ion modes, with high reproducibility. In addition, the above-mentioned special properties of MBT allow its employment for imaging mass spectrometry. In the present work, images of brain and liver tissues showing different lipid species are presented, demonstrating the advantages of the employment of MBT.

Distribution of lipids in human brain

The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Over the last decades, some lipids in the CNS have been identified as intracellular signalers, while others are known to act as neuromodulators of neurotransmission through binding to specific receptors. Neurotransmitters of lipidic nature, currently known as neurolipids, are synthesized during the metabolism of phospholipid precursors present in cell membranes. Therefore, the anatomical identification of each of the different lipid species in human CNS by imaging mass spectrometry (IMS), in association with other biochemical techniques with spatial resolution, can increase our knowledge on the precise metabolic routes that synthesize these neurolipids and their localization. The present study shows the lipid distribution obtained by MALDI-TOF IMS in human frontal cortex, hippocampus, and striatal area, together with functional autoradiography of cannabinoid and LPA receptors. The combined application of these methods to postmortem human brain samples may be envisioned as critical to further understand neurological diseases, in general, and particularly, the neurodegeneration that accompanies Alzheimer’s disease.

Crystallization and preliminary crystallographic analysis of merohedrally twinned crystals of MJ0729, a CBS-domain protein from Methanococcus jannaschii

CBS domains are small protein motifs, usually associated in tandem, that are implicated in binding to adenosyl groups. Several genetic diseases in humans have been associated with mutations in CBS sequences, which has made them very promising targets for rational drug design. Trigonal crystals of the CBS-domain protein MJ0729 from Methanococcus jannaschii were grown by the vapour-diffusion method at acidic pH. Preliminary analysis of nine X-ray diffraction data sets using Yeates statistics and Britton plots showed that slight variation in the pH as well as in the buffer used in the crystallization experiments led to crystals with different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning.

Purification, crystallization and preliminary X-ray diffraction analysis of the CBS-domain pair from the Methanococcus jannaschii protein MJ0100.

CBS domains are small protein motifs consisting of a three-stranded beta-sheet and two alpha-helices that are present in proteins of all kingdoms of life and in proteins with completely different functions. Several genetic diseases in humans have been associated with mutations in their sequence, which has made them promising targets for rational drug design. The C-terminal domain of the Methanococcus jannaschii protein MJ0100 includes a CBS-domain pair and has been overexpressed, purified and crystallized. Crystals of selenomethionine-substituted (SeMet) protein were also grown. The space group of both the native and SeMet crystals was determined to be orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 80.9, b = 119.5, c = 173.3 A. Preliminary analysis of the X-ray data indicated that there were eight molecules per asymmetric unit in both cases.

Descifrando la anatomía molecular de un tejido mediante análisis lipidómico por espectrometría de masas

Resumen La lipidomica esta emergiendo como una ciencia imprescindible para la comprension del funcionamiento de celulas y tejidos, dado que complementa el conocimiento obtenido mediante la genomica y la proteomica. La caracterizacion de los lipidos que componen una muestra biologica es una tarea compleja, debido a su enorme variedad y variabilidad, y por tanto necesita del empleo de tecnicas de alto rendimiento para su consecucion. La espectrometria de masas esta demostrando ser la tecnica ideal para llevar a cabo dicha tarea, especialmente desde el desarrollo de la imagen por espectrometria de masas. En el presente trabajo se presentan los resultados del empleo de una nueva matriz, 2-mercaptobenzotiazol, en el analisis lipidomico de extractos de higado quiescente y regenerante de roedor mediante espectrometria de masas MALDI-TOF. La buena reproducibilidad entre individuos, permite la observacion de diferencias en las especies lipidicas mayoritarias del higado normal y el afectado de esteatosis. Se muestran ademas imagenes de la disposicion anatomica de ciertas moleculas en el cerebro de rata, obtenidas representando la distribucion de las concentraciones de diversas especies de lipidos a lo largo de los cortes de tejido. Por ultimo, se discute la aplicacion de estas tecnicas a la investigacion experimental y clinica.