Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of dextran and dextrin derivatives

Application of matrix-assisted laser desorption/ionization (MALDI) to the analysis of dextran and dextrin derivatives, specifically glucose saccharides, by time-of-flight (TOF) mass spectrometry has been reported. MALDI-TOF analysis was carried out on alpha-, beta- and gamma-cyclodextrin, two O-methylated-beta-cyclodextrins of differing degrees of substitution (DS) and dextrans (a linear glucose saccharide), as pure and doped solutions and as mixtures of two or more of these. Doping was carried out with trace amounts of inorganic salts. The purpose of the analysis of the cyclodextrins was to determine whether they would form inclusion complexes with the various cations added, or whether less specific cation addition/exchange was occurring either prior to desorption or in the gas phase.     

Accuracy and validity of stereology as a quantitative method for assessment of human temporal lobe volumes acquired by magnetic resonance imaging

The object of this study was to compare the accuracy and validity of stereology as a method for determining whole temporal lobe volume with the more established technique of semi-automated thresholding and tracing. Ten, fixed, post-mortem human brains, were imaged using a three dimensional (3D) acquisition protocol. The volume of the left temporal lobe, dissected from each brain, was determined by fluid displacement. Each volume was compared to measurements obtained from magnetic resonance images (MRI) of the post-mortem brain using each of the two segmentation methods. Post-acquisition processing was performed using MEASURE software. Three investigators performed each measurement three times using each method, yielding a total of 180 measurements. Stereology took, on average, half the time of thresholding/tracing. Using a clinically acceptable variation for 95% of repeat measures; both intra-observer and inter-observer variation were acceptable for each technique. However, validity, as demonstrated by graphs of agreement against water displacement showed that the "limits of agreement" using stereology were within the acceptable range, while those using the thresholding/tracing technique were not. Quantitative estimates of variation and a graphical representation of the limits of agreement show that stereology is at least as precise as the thresholding/tracing method but is superior in terms of speed and validity. This has broad implications for published estimates of brain region volumes in human diseases such as epilepsy, dementia and other neurodegenerative disorders.     

Engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents

Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.     

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

Structural elucidation studies of erythromycins by electrospray tandem mass spectrometry

Erythromycin A (EryA) was studied by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for the structural elucidation of novel erythromycins developed by biological synthetic methods. Skimmer dissociation along with sequential mass spectrometry studies (up to MS5) have been employed in this study. In the low-resolution MS/MS analysis of the polyketides, there are several fragment ions that are easily assigned to various neutral losses. These have all been confirmed by accurate-mass measurements. There is also a series of peaks due to ring opening and fragmentation that can only be assigned by high-resolution MSn analysis. Further experiments were performed in deuterated media (D2O/CD3OD 50%) which, along with the high-resolution MSn of erythromycin analogues, has enabled us to identify some of the steps in the ring fragmentation, particularly the loss of the polyketide starter acid. This is an essential step for determining structural alterations in the novel polyketides, but further labelling experiments and studies on more erythromycin analogues are required before the complete fragmentation pathway can be confirmed.     

Antibody-combining sites

The antibody repertoire is very large with at least 10⁹ different antibody specificities, yet there are currently only 800 variable-region sequences known and < 23 Fab structures deposited with the Brookhaven Protein Data Bank. To engineer the antibody-combining site rationally, we need to define the rules that govern antibody structure. To understand the process of antibody-antigen recognition, we need not only to predict complementary determining regions accurately, but to simulate accurately the interaction of antibody with antigen. We have made progress in the modeling of antibody-combining sites and in the simulation of antibody complex formation. The combination of these approaches will allow us to extend the natural limits of antibody-combining sites in a more rational manner.