Simple deprotection of acetal type protecting groups under neutral conditions

The hallmarks of Alzheimer's disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Aβ-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network

Changes in salivary analytes in canine parvovirus : A high-resolution quantitative proteomic study

The present study evaluated the changes in salivary proteome in parvoviral enteritis (PVE) in dogs through a high-throughput quantitative proteomic analysis. Saliva samples from healthy dogs and dogs with severe parvovirosis that survived or perished due to the disease were analysed and compared by Tandem Mass Tags (TMT) analysis. Proteomic analysis quantified 1516 peptides, and 287 (corresponding to 190 proteins) showed significantly different abundances between studied groups. Ten proteins were observed to change significantly between dogs that survived or perished due to PVE. Bioinformatics' analysis revealed that saliva reflects the involvement of different pathways in PVE such as catalytic activity and binding, and indicates antimicrobial humoral response as a pathway with a major role in the development of the disease. These results indicate that saliva proteins reflect physiopathological changes that occur in PVE and could be a potential source of biomarkers for this disease

Regional Specializations of the PAZ Proteomes Derived from Mouse Hippocampus, Olfactory Bulb and Cerebellum

Neurotransmitter release as well as structural and functional dynamics at the presynaptic active zone (PAZ) comprising synaptic vesicles attached to the presynaptic plasma membrane are mediated and controlled by its proteinaceous components. Here we describe a novel experimental design to immunopurify the native PAZ-complex from individual mouse brain regions such as olfactory bulb, hippocampus, and cerebellum with high purity that is essential for comparing their proteome composition. Interestingly, quantitative immunodetection demonstrates significant differences in the abundance of prominent calcium-dependent PAZ constituents. Furthermore, we characterized the proteomes of the immunoisolated PAZ derived from the three brain regions by mass spectrometry. The proteomes of the release sites from the respective regions exhibited remarkable differences in the abundance of a large variety of PAZ constituents involved in various functional aspects of the release sites such as calcium homeostasis, synaptic plasticity and neurogenesis. On the one hand, our data support an identical core architecture of the PAZ for all brain regions and, on the other hand, demonstrate that the proteinaceous composition of their presynaptic active zones vary, suggesting that changes in abundance of individual proteins strengthen the ability of the release sites to adapt to specific functional requirements

APP Is a Context-Sensitive Regulator of the Hippocampal Presynaptic Active Zone

The hallmarks of Alzheimer's disease (AD) are characterized by cognitive decline and behavioral changes. The most prominent brain region affected by the progression of AD is the hippocampal formation. The pathogenesis involves a successive loss of hippocampal neurons accompanied by a decline in learning and memory consolidation mainly attributed to an accumulation of senile plaques. The amyloid precursor protein (APP) has been identified as precursor of Abeta-peptides, the main constituents of senile plaques. Until now, little is known about the physiological function of APP within the central nervous system. The allocation of APP to the proteome of the highly dynamic presynaptic active zone (PAZ) highlights APP as a yet unknown player in neuronal communication and signaling. In this study, we analyze the impact of APP deletion on the hippocampal PAZ proteome. The native hippocampal PAZ derived from APP mouse mutants (APP-KOs and NexCreAPP/APLP2-cDKOs) was isolated by subcellular fractionation and immunopurification. Subsequently, an isobaric labeling was performed using TMT6 for protein identification and quantification by high-resolution mass spectrometry. We combine bioinformatics tools and biochemical approaches to address the proteomics dataset and to understand the role of individual proteins. The impact of APP deletion on the hippocampal PAZ proteome was visualized by creating protein-protein interaction (PPI) networks that incorporated APP into the synaptic vesicle cycle, cytoskeletal organization, and calcium-homeostasis. The combination of subcellular fractionation, immunopurification, proteomic analysis, and bioinformatics allowed us to identify APP as structural and functional regulator in a context-sensitive manner within the hippocampal active zone network

Relative abundance of proteins mapped to the functional subnetwork of calcium homeostasis.

<p>A Impact of APP deletion. B Impact of the NexCre-cDKO. Change in abundance of more than ±10% is reflected by increasing sizes of nodes. The color code corresponds to the degree of up- (magenta) and downregulation (green). Nodes in yellow represent proteins with changes in abundance of less than ±10%. Abbreviations are the respective gene names of individual proteins as given in UniProt database and in the supplementary information <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004832#pcbi.1004832.s001" target="_blank">S1 Table</a>.</p

Relative abundance of proteins mapped to the functional subnetwork of the cytoskeleton organization.

<p>A Impact of APP deletion. B Impact of the NexCre-cDKO. Change in abundance of more than ±10% is reflected by increasing sizes of nodes. The color code corresponds to the degree of up- (magenta) and downregulation (green). Nodes in yellow represent proteins with changes in abundance of less than ±10%. Abbreviations are the respective gene names of individual proteins as given in UniProt database and in the supplementary information <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004832#pcbi.1004832.s001" target="_blank">S1 Table</a>.</p

Scheme illustrating the regulatory role of APP in a context-sensitive manner at the hippocampal PAZ. R, regulator; M, mediator, C, central player.

<p>Color code: magenta, upregulation (in APP-mutants); green, downregulation (in APP-mutants); yellow, unaltered.</p

The interactome of the native hippocampal PAZ core proteome.

<p>A Proteins grouped according to their localization (localization layout). B Community structure layout (function) of the network. The size of the rings corresponds to the respective number of proteins. The color code corresponds to the pie chart diagram (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004832#pcbi.1004832.g001" target="_blank">Fig 1E</a>). C Impact of APP deletion on relative protein abundance mapped according to their localizations. D Impact of the APP-KO on relative protein abundance mapped according to the community structure. E Impact of the NexCre-cDKO on relative protein abundance mapped according to their localizations. F Impact of the NexCre-cDKO on relative protein abundance mapped according to the community structure. Change in abundance of more than ±10% is reflected by increasing sizes of nodes. The color code corresponds to the degree of up- (magenta) and downregulation (green). Nodes in yellow represent proteins with changes in abundance of less than ±10%. Each node (dot in the rings) within this network represents a protein and each edge (connection) represents a reported physical interaction between two proteins. Edges are bundled for clarity.</p

Relative abundance of proteins mapped to the functional subnetwork of the synaptic vesicle cycle.

<p>A Impact of APP deletion. B Impact of the NexCre-cDKO. Change in abundance of more than ±10% is reflected by increasing sizes of nodes. The color code corresponds to the degree of up- (magenta) and downregulation (green). Nodes in yellow represent proteins with changes in abundance of less than ±10%. Abbreviations are the respective gene names of individual proteins as given in UniProt database and in the supplementary information <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004832#pcbi.1004832.s001" target="_blank">S1 Table</a>.</p

Overview of the experimental design.

<p>A Workflow of subcellular fractionation and immunopurification of the native hippocampal PAZ. B Experimental outline of isobaric labeling of peptides with TMT⁶ and MS analysis by nano-high-pressure liquid chromatography (nHPLC-ESI). C Example of peptide signals (m/z) for the six reporter groups. D Differences in protein abundance of hippocampal PAZ constituents between APP-mutant and control. E Pie chart diagram of proteins attributed to the PAZ. F Scheme of a PPI network illustrating proteins (exemplarily designated as A-K) as nodes and edge betweeness. The thickness of the connections represents the importance of the respective edges for information flow within the network (edge betweenness). Change in abundance of more than ±10% is reflected by increasing sizes of nodes. The color code corresponds to the degree of up- (magenta) and downregulation (green). Nodes in yellow represent proteins with changes in abundance of less than ±10%. UF, upper fractions; LF, lower fractions; IP, immunopurification, MB, magnetic bead; PM, plasma membrane; SV, synaptic vesicle, SC, signaling cascade; CS, cytoskeleton; ME, metabolic enzymes; MI, mitochondria; O, others.</p