Characterization of N-terminal processing of group VIA phospholipase A<Subscript>2</Subscript> and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.     

Structure determination of Ls-threonine by neutron diffraction

The structure of the aminoacid, L_s-threonine [NH ₃ ⁺ CH(CHOHCH₃)COO^?], space groupP2₁2₁2₁,a=13.630(5),b=7.753(1),c=5.162(2) Å,z=4, has been determined from neutron diffraction data using direct methods. The intensities of 1148 neutron Bragg reflections were measured from a single crystal. The structural parameters were refined by the method of least squares using anisotropic temperature factors. The finalR(F ²) is 0.068. The structure was also refined from the x-ray data of Shoemakeret al (1950J. Am. Chem. Soc. 72 2328); there is good agreement between the two sets of heavy atom parameters. The parameters of hydrogen atoms are of course more precisely determined in our neutron study. The molecular conformation and the hydrogen bonding scheme are discussed. Weighted average values of bond distances and angles from 14 aminoacid structures with ionized carboxylic groups studied by neutron diffraction at Brookheven and Trombay are also presented.     

Phase transition in triglycine family of hydrogen bonded ferroelectrics: An interpretation based on structural studies

Using the crystal structure, a comprehensive interpretation of the origin of ferroelectricity in the hydrogen bonded triglycine family of crystals is given. Our detailed analysis showed that the instability of nitrogen double well potential plays a driving role in the mechanism of the ferroelectric transitions in these crystals.     

Low-temperature neutron diffraction study of La<Subscript>0.95</Subscript>Nd<Subscript>0.05</Subscript>CrO<Subscript>3</Subscript>

We have synthesized polycrystalline La_0.95Nd_0.05CrO₃ sample by doping the La-site of LaCrO₃ with Nd and its magnetic properties have been studied using DC magnetization and neutron diffraction techniques. DC magnetization study shows a paramagnetic to a weak ferromagnetic-like transition at ∼295 K followed by signatures of a spin reorientation phenomenon at 233 and 166 K and, finally a transition to an antiferromagnetic-like phase at ∼21 K. Low-temperature neutron diffraction measurements confirm a weak ferrimagnetic ordering of Cr³⁺ moments at all temperatures below 295 K.     

Structural changes during the unfolding of Bovine serum albumin in the presence of urea: A small-angle neutron scattering study

The native form of serum albumin is the most important soluble protein in the body plasma. In order to investigate the structural changes of Bovine serum albumin (BSA) during its unfolding in the presence of urea, a small-angle neutron scattering (SANS) study was performed. The scattering curves of dilute solutions of BSA with different concentrations of urea in D₂O at pH 7.2 ± 0.2 were measured at room temperature. The scattering profile was fitted to a prolate ellipsoidal shape (a, b, b) of the protein witha = 52.2 Å andb = 24.2 Å. The change in the dimensions of the protein as it unfolds was found to be anisotropic. The radius of gyration of the compact form of the protein in solution decreased as the urea concentration was increased.     

Quantification of distinct molecular species of the 2-lyso metabolite of platelet-activating factor by gas chromatography-negative-ion chemical ionization mass spectrometry.

The biological activity of platelet-activating factor (PAF) is comprised by a few molecular species of phosphatidylcholine which contain a fatty alcohol connected by an ether linkage to the sn-1 position of the glycerol backbone and an acetate ester at the sn-2 position. The various molecular species of PAF differ in chain length and degree of unsaturation in the fatty alcohol residue side-chain. PAF is rapidly hydrolyzed to lyso-PAF by an acetylhydrolase enzyme which is quite active in a number of cells that synthesize PAF. We describe a method for quantitation of lyso-PAF which involves conversion to its propionate derivative in the presence of an internal standard (deuterium-labelled PAF), digestion to the diglyceride with Bacillus cereus phospholipase C, conversion to the pentafluorobenzoate derivative and capillary column gas chromatographic-negative-ion methane chemical ionization mass spectrometric analysis. Distinct molecular species of lyso-PAF can be individually quantitated at levels of 1 ng or less. These methods are applied to the demonstration of lyso-PAF accumulation in renal tissue from transplanted allografts undergoing acute rejection, in renal tissue from kidneys subjected to cold storage and autotransplantation, and in intestinal mucosa subjected to warm ischemia and reperfusion.