Development of an Online Cell-Based Bioactivity Screening Method by Coupling Liquid Chromatography to Flow Cytometry with Parallel Mass Spectrometry

This study describes a new platform for the fast and efficient functional screening for bioactive compounds in complex natural mixtures using a cell-based assay. The platform combines reversed-phase liquid chromatography (LC) with online flow cytometry (FC) and mass spectrometry (MS). As a model (an example or proof-of-concept study) we have used a functional calcium-flux assay in human neuroblastoma SH-SY5Y cells stably overexpressing the α-7 nicotinic acetylcholine receptor (α7-nAChR), a potential therapeutic target for central nervous system (CNS) related diseases. We have designed the coupled LC–FC system employing the neuroblastoma cells followed by analytical and pharmacological evaluation of the hyphenated setup in agonist and mixed antagonist–agonist assay modes. Using standard receptor ligands we have validated pharmacological responses and standardized good assay quality parameters. The applicability of the screening system was evaluated by analysis of various types of natural samples, such as a tobacco plant extract (in agonist assay mode) and snake venoms (in mixed antagonist–agonist assay mode). The bioactivity responses were correlated directly to the respective accurate masses of the compounds. Using simultaneous functional agonist and antagonist responses nicotine and known neurotoxins were detected from tobacco extract and snake venoms, respectively. Thus, the developed analytical screening technique represents a new tool for rapid measurement of functional cell-based responses and parallel separation and identification of compounds in complex mixtures targeting the α7-nAChR. It is anticipated that other fast-response cell-based assays (e.g., other ion flux assays) can be incorporated in this analytical setup

Additional file 5: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Figure S4. Clustering analysis of experimental groups and individual cases. Clustering analysis and heat maps of the different experimental groups (A) and individual cases (B) based on proteins with a significant difference (ANOVA, p < 0.05) in expression between any of the groups. (TIF 709 kb

Additional file 1: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Figure S1. Coomassie blue staining of the SDS PAGE gels containing the microdissected tissue lysates. (TIF 478 kb

Additional file 7: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Figure S6. Protein expression of males versus females. Quantitative data on several CAA selective data was plotted with males represented as triangles and females as dots. No clear relationship between gender and protein abundance was observed. (TIF 24739 kb

Additional file 6: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Figure S5. Protein expression of CAA case #5 relative to the experimental groups and individual cases. (A) On the left the expression profile of case #5 compared to the average expression profile of the control group (2nd row), AD group (3rd row) and the CAA group (4th row). Green, expression below the overall mean; red, above the overall mean. The expression profile of case #5 is largely similar to that of the control groups but some proteins show a similar expression as in the AD and/or CAA groups. (B) Expression values (LFQ values) of several CAA specific proteins identified in this study with case #5 indicated as empty triangle pointing down. Case #5 does not differ from the CAA group in these markers. (TIF 1835 kb

Additional file 8: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Figure S7. Immunoreactivity for COL6A2 is equally present in leptomeningeal vessels in control, AD and CAA tissue. (TIF 3794 kb

Additional file 2: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Figure S2. Total protein fluorescent signal from blots used for immunoblot analysis. Total protein load was visualized using a chemidoc EZ (Bio-Rad) after electroblotting and used to obtain densitometric values which were then used to normalize for total protein input. (TIF 553 kb

Additional file 4: of Proteomics analysis identifies new markers associated with capillary cerebral amyloid angiopathy in Alzheimer’s disease

Table S1. Complete dataset, containing log2 transformed quantitative values (LFQ values) of all quantified proteins per individual case. (XLSX 665 kb

Proteomic Analysis of Gliosomes from Mouse Brain: Identification and Investigation of Glial Membrane Proteins

Astrocytes are being increasingly recognized as crucial contributors to neuronal function at synapses, axons, and somas. Reliable methods that can provide insight into astrocyte proteins at the neuron–astrocyte functional interface are highly desirable. Here, we conducted a mass spectrometry analysis of Percoll gradient-isolated gliosomes, a viable preparation of glial subcellular particles often used to study mechanisms of astrocytic transmitter uptake and release and their regulation. Gliosomes were compared with synaptosomes, a preparation containing the neurotransmitter release machinery, and, accordingly, synaptosomes were enriched for proteins involved in synaptic vesicle-mediated transport. Interestingly, gliosome preparations were found to be enriched for different classes of known astrocyte proteins, such as VAMP3 (involved in astrocyte exocytosis), Ezrin (perisynaptic astrocyte cytoskeletal protein), and Basigin (astrocyte membrane glycoprotein), as well as for G-protein-mediated signaling proteins. Mass spectrometry data are available via ProteomeXchange with the identifier PXD001375. Together, these data provide the first detailed description of the gliosome proteome and show that gliosomes can be a useful preparation to study glial membrane proteins and associated processes

Proteomic Analysis of Gliosomes from Mouse Brain: Identification and Investigation of Glial Membrane Proteins

Astrocytes are being increasingly recognized as crucial contributors to neuronal function at synapses, axons, and somas. Reliable methods that can provide insight into astrocyte proteins at the neuron–astrocyte functional interface are highly desirable. Here, we conducted a mass spectrometry analysis of Percoll gradient-isolated gliosomes, a viable preparation of glial subcellular particles often used to study mechanisms of astrocytic transmitter uptake and release and their regulation. Gliosomes were compared with synaptosomes, a preparation containing the neurotransmitter release machinery, and, accordingly, synaptosomes were enriched for proteins involved in synaptic vesicle-mediated transport. Interestingly, gliosome preparations were found to be enriched for different classes of known astrocyte proteins, such as VAMP3 (involved in astrocyte exocytosis), Ezrin (perisynaptic astrocyte cytoskeletal protein), and Basigin (astrocyte membrane glycoprotein), as well as for G-protein-mediated signaling proteins. Mass spectrometry data are available via ProteomeXchange with the identifier PXD001375. Together, these data provide the first detailed description of the gliosome proteome and show that gliosomes can be a useful preparation to study glial membrane proteins and associated processes