Laser-induced
fluorescence detection (LIF) is a powerful tool for
the quantitative analysis of fluorescent molecules, widely used in
glycan analysis with fluorophore labeled carbohydrates where each
species has a common response factor. Electrospray ionization mass
spectrometry (ESI-MS), on the other hand, while revealing important
structural information about individual analytes, generally can have
different response factors for different species. For simpler and
improved quantitation with ESI-MS, laser-induced fluorescent images
were collected at the Taylor cone of the electrospray interface, enabling
simultaneous and robust optical (quantitative) and MS (qualitative)
detection of fluorophore labeled sugars. The performance of this universally
applicable, interface design independent imaging laser-induced fluorescent
(iLIF) system was demonstrated using capillary electrophoresis (CE)-ESI-MS
in the analysis of aminopyrene-trisulfonate labeled linear maltooligosaccharides
and branched glycans from human immunoglobulin. The limit of detection
(LOD) of the iLIF system was in this case 40 attomole. The intra-
and interday quantitative (peak area) reproducibilities of the system
(RSD) were 4.15% and 6.79%, respectively.
Alzheimer’s disease (AD) and neocortical Lewy body disease (LBD) are the most common neurodegenerative dementias, with no available curative treatment. Elucidating pathomechanism and identifying novel therapeutic targets are of paramount importance. Lemur tyrosine kinase 2 (LMTK2) is involved in several physiological and pathological cellular processes. Herewith a neuropathological characterization is presented in AD and neocortical LBD samples using chromogenic and fluorescent LMTK2 immunohistochemistry on post-mortem brain tissues and compared them to age-matched controls (CNTs). LMTK2 immunopositivity was limited to the neuronal cytoplasm. Neurons, including tau-positive tangle-bearing ones, showed decreased chromogenic and immunofluorescent labelling in AD in every cortical layer compared to CNT and neocortical LBD. Digital image analysis was performed to measure the average immunopositivity of groups. Mean grey values were calculated for each group after measuring the grey scale LMTK2 signal intensity of each individual neuron. There was significant difference between the mean grey values of CNT vs. AD and neocortical LBD vs. AD. The moderate decrease in neocortical LBD suggests the effect of coexisting AD pathology. We provide neuropathological evidence on decreased neuronal LMTK2 immunolabelling in AD, with implications for pathogenesis.
We present the application of a smartphone anatomy based technology in the field of liquid phase bioseparations, particularly in capillary electrophoresis. A simple capillary electrophoresis system was built with LED induced fluorescence detection and a credit card sized minicomputer to prove the concept of real time fluorescent imaging (zone adjustable time-lapse fluorescence image processor) and separation controller. The system was evaluated by analyzing under- and overloaded aminopyrenetrisulfonate (APTS)-labeled oligosaccharide samples. The open source software based image processing tool allowed undistorted signal modulation (reprocessing) if the signal was inappropriate for the actual detection system settings (too low or too high). The novel smart detection tool for fluorescently labeled biomolecules greatly expands dynamic range and enables retrospective correction for injections with unsuitable signal levels without the necessity to repeat the analysis.
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