ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711.

Amyloid-ß (Aß) deposition in the brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD). We have previously identified amyloid precursor protein (APP)669-711 (a.k.a. Aß(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS). Furthermore, we found that the level of a composite biomarker, i.e., a combination of APP669-711/Aß1-42 ratio and Aß1-40/Aß1-42 ratio in human plasma, correlates with the amyloid PET status of AD patients. However, the production mechanism of APP669-711 has remained unclear. Using in vitro and in vivo assays, we identified A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) as a responsible enzyme for APP669-711 production. ADAMTS4 cleaves APP directly to generate the C-terminal stub c102, which is subsequently proteolyzed by γ-secretase to release APP669-711. Genetic knockout of ADAMTS4 reduced the production of endogenous APP669-711 by 30% to 40% in cultured cells as well as mouse plasma, irrespectively of Aß levels. Finally, we found that the endogenous murine APP669-711/Aß1-42 ratio was increased in aged AD model mice, which shows Aß deposition as observed in human patients. These data suggest that ADAMTS4 is involved in the production of APP669-711, and a plasma biomarker determined by IP-MALDI-MS can be used to estimate the level of Aß deposition in the brain of mouse models.

Identifying Double Bond Positions in Phospholipids Using Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry Based on Oxygen Attachment Dissociation.

Lipids, a class of biomolecules, play a significant role in the physiological system. In this study, gas-phase hydroxyl radicals (OH·) and atomic oxygens (O) were introduced into the collision cell of a triple quadruple mass spectrometer (TQ-MS) to determine the positions of the double bond in unsaturated phospholipids. A microwave-driven compact plasma generator was used as the OH·/O source. The reaction between OH·/O and the precursor ions passing through the collision cell generates product ions that correspond to the double bond positions in the fatty acyl chain. This double bond position specific fragmentation process initiated by the attachment of OH·/O to the double bond of a fatty acyl chain is a characteristic of oxygen attachment dissociation (OAD). A TQ-MS incorporating OAD, in combination with liquid chromatography, permitted a high throughput analysis of the double bond positions in complex biomolecules. It is important to know the precise position of double bonds in lipids, since these molecules can have widely different functionalities based on the position of the double bonds. The assignment of double bond positions in a mixture of eight standard samples of phosphatidylcholines (phospholipids with choline head groups) with multiple saturated fatty acyl chains attached was successfully demonstrated.

Inkjet automated single cells and matrices printing system for matrix-assisted laser desorption/ionization mass spectrometry.

The ability of single or several cells introduction onto substrate simply would be a useful tool for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this study, we aimed to establish a sample introduction method for pattering cells to the substrate by inkjet technology. Inkjet driving, substrate surface and relative humidity were optimized for single or several cells introduction. Single type cell solution and MALDI matrix solution were automatically printed onto ITO glass substrate which was hydrophobic modified under humidity controlled condition. Then the substrate was inserted to MALDI-MS and cells sample solution provided several peaks from phospholipids. The inkjet technique enables us to print single and subcellular on the substrate with the range of a few hundred micrometers. This diameter would be useful for targeting by laser of MALDI-MS. Our technique provides a new platform for MALDI-MS analysis in single or several cells to get a wide information from one sample.