Decoding CPK/SnRK Superfamily Kinase Client Signaling Networks Using Peptide Library and Mass Spectrometry.

Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in , the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways.

Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome.

Substance use disorders are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids.

Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome.

Substance use disorders (SUDs) are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids.

The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface.

Simple, Single-Shot Phosphoproteomic Analysis of Heat-Stable Tau Identifies Age-Related Changes in pS235- and pS396-Tau Levels in Non-human Primates.

Age is the most significant risk factor for Alzheimer's disease (AD), and understanding its role in specific aspects of AD pathology will be critical for therapeutic development. Neurofibrillary tangles composed of hyperphosphorylated tau are a quintessential hallmark of AD. To study age-related changes in tau phosphorylation, we developed a simple, antibody-free approach for single shot analysis of tau phosphorylation across the entire protein by liquid-chromatography tandem mass spectrometry.

Sex Differences in the Ventral Tegmental Area and Nucleus Accumbens Proteome at Baseline and Following Nicotine Exposure.

Sex differences in behaviors relevant to nicotine addiction have been observed in rodent models and human subjects. Behavioral, imaging, and epidemiological studies also suggest underlying sex differences in mesolimbic dopamine signaling pathways. In this study we evaluated the proteome in the ventral tegmental area (VTA) and nucleus accumbens (NAc) shell in male and female mice.

Cocaine self-administration induces sex-dependent protein expression in the nucleus accumbens.

Substance use disorder (SUD) is a chronic neuropsychiatric condition characterized by long-lasting alterations in the neural circuitry regulating reward and motivation. Substantial work has focused on characterizing the molecular substrates that underlie these persistent changes in neural function and behavior. However, this work has overwhelmingly focused on male subjects, despite mounting clinical and preclinical evidence that females demonstrate dissimilar progression to SUD and responsivity to stimulant drugs of abuse, such as cocaine.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments.

Granulocyte-Colony Stimulating Factor Reduces Cocaine-Seeking and Downregulates Glutamatergic Synaptic Proteins in Medial Prefrontal Cortex.

Psychostimulant use disorder is a major public health issue, and despite the scope of the problem there are currently no Food and Drug Administration (FDA)-approved treatments. There would be tremendous utility in development of a treatment that could help patients both achieve and maintain abstinence. Previous work from our group has identified granulocyte-colony stimulating factor (G-CSF) as a neuroactive cytokine that alters behavioral response to cocaine, increases synaptic dopamine release, and enhances cognitive flexibility.

Differential Protein Expression in Striatal D1- and D2-Dopamine Receptor-Expressing Medium Spiny Neurons.

Many neurological disorders and diseases including drug addiction are associated with specific neuronal cell types in the brain. The striatum, a region that plays a critically important role in the development of addictive drug-related behavior, provides a good example of the cellular heterogeneity challenges associated with analyses of specific neuronal cell types. Such studies are needed to identify the adaptive changes in neuroproteomic signaling that occur in response to diseases such as addiction.

Mass Spectrometry-Based Identification of Phospho-Tyr in Plant Proteomics.

O-Phosphorylation (phosphorylation of the hydroxyl-group of S, T, and Y residues) is among the first described and most thoroughly studied posttranslational modification (PTM). Y-Phosphorylation, catalyzed by Y-kinases, is a key step in both signal transduction and regulation of enzymatic activity in mammalian systems. Canonical Y-kinase sequences are absent from plant genomes/kinomes, often leading to the assumption that plant cells lack -phospho-l-tyrosine (pY).

Proteomic characterization of low molecular weight allergens and putative allergen proteins in lentil (Lens culinaris) cultivars of Bangladesh.

Here, we present the proteome profiling of low-molecular weight (<50 kDa) proteins of seven different lentil cultivars developed by Bangladesh Agricultural Research Institute. A total of 2873 peptides corresponding to 180 unique proteins were identified wherein >24% of them were described lentil allergens. Comparative relative quantitation showed differences in protein abundance of major allergen proteins such as Len c 1.

Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD).

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD.

Cell-Type-Specific Proteomics: A Neuroscience Perspective.

Cell-type-specific analysis has become a major focus for many investigators in the field of neuroscience, particularly because of the large number of different cell populations found in brain tissue that play roles in a variety of developmental and behavioral disorders. However, isolation of these specific cell types can be challenging due to their nonuniformity and complex projections to different brain regions. Moreover, many analytical techniques used for protein detection and quantitation remain insensitive to the low amounts of protein extracted from specific cell populations.

ProteomicsBrowser: MS/proteomics data visualization and investigation.

Summary: Large-scale, quantitative proteomics data are being generated at ever increasing rates by high-throughput, mass spectrometry technologies. However, due to the complexity of these large datasets as well as the increasing numbers of post-translational modifications (PTMs) that are being identified, developing effective methods for proteomic visualization has been challenging. ProteomicsBrowser was designed to meet this need for comprehensive data visualization.

Evaluation of the Phosphoproteome of Mouse Alpha 4/Beta 2-Containing Nicotinic Acetylcholine Receptors In Vitro and In Vivo.

Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit.

Granulocyte-Colony-Stimulating Factor Alters the Proteomic Landscape of the Ventral Tegmental Area.

Cocaine addiction is characterized by aberrant plasticity of the mesolimbic dopamine circuit, leading to dysregulation of motivation to seek and take drug. Despite the significant toll that cocaine use disorder exacts on society, there are currently no available pharmacotherapies. We have recently identified granulocyte-colony stimulating factor (G-CSF) as a soluble cytokine that alters the behavioral response to cocaine and which increases dopamine release from the ventral tegmental area (VTA).

Isoform-Level Interpretation of High-Throughput Proteomics Data Enabled by Deep Integration with RNA-seq.

Cellular control of gene expression is a complex process that is subject to multiple levels of regulation, but ultimately it is the protein produced that determines the biosynthetic state of the cell. One way that a cell can regulate the protein output from each gene is by expressing alternate isoforms with distinct amino acid sequences. These isoforms may exhibit differences in localization and binding interactions that can have profound functional implications.

Absolute Quantitation of Plant Proteins.

Among targeted proteomic techniques, AQUA-MRM is considered as one of the most reliable for accurate protein quantitation. This method displays high sensitivity, specificity, and reproducibility compared to many common biochemical techniques by coupling the use of unique, heavy-labeled peptide standards and triple-quadrupole mass spectrometry. However, there are several important steps that are required for successful development and validation of a robust AQUA-MRM assay.

In Vivo Quantitative Monitoring of Subunit Stoichiometry for Metabolic Complexes.

Metabolic pathways often employ assemblies of individual enzymes to facilitate substrate channeling to improve thermodynamic efficiency and confer pathway directionality. It is often assumed that subunits to multienzyme complexes are coregulated and accumulate at fixed levels in vivo, reflecting complex stoichiometry. Such assumptions can be experimentally tested using modern tandem mass spectrometry, and herein we describe such an approach applied toward an important metabolic complex.

Luxurious Nitrogen Fertilization of Two Sugar Cane Genotypes Contrasting for Lignin Composition Causes Changes in the Stem Proteome Related to Carbon, Nitrogen, and Oxidant Metabolism but Does Not Alter Lignin Content.

Sugar cane is an important crop for sugar and biofuel production. Its lignocellulosic biomass represents a promising option as feedstock for second-generation ethanol production. Nitrogen fertilization can affect differently tissues and its biopolymers, including the cell-wall polysaccharides and lignin.

Comparative proteomic analysis provides insight into the biological role of protein phosphatase inhibitor-2 from Arabidopsis.

Unlabelled: Protein phosphatase inhibitor-2 (PPI-2) is a conserved eukaryotic effector protein that inhibits type one protein phosphatases (TOPP). A transfer-DNA knockdown of AtPPI-2 resulted in stunted growth in both vegetative and reproductive phases of Arabidopsis development. At the cellular level, AtPPI-2 knockdown had 35 to 40% smaller cells in developing roots and leaves.

Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins.

14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms.

Multisite phosphorylation of 14-3-3 proteins by calcium-dependent protein kinases.

Plant 14-3-3 proteins are phosphorylated at multiple sites in vivo; however, the protein kinase(s) responsible are unknown. Of the 34 CPK (calcium-dependent protein kinase) paralogues in Arabidopsis thaliana, three (CPK1, CPK24 and CPK28) contain a canonical 14-3-3-binding motif. These three, in addition to CPK3, CPK6 and CPK8, were tested for activity against recombinant 14-3-3 proteins χ and ε.