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. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

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. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based quantitative proteomics, along with a data-independent acquisition analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveal cocaine and morphine differentially alter diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between the phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome. The proteomics data in this study are available via ProteomeXchange with identifier PXD042043.

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. Here, we investigate the role of G-CSF in affecting extinction and reinstatement of cocaine-seeking and perform detailed characterization of its proteomic effects in multiple limbic substructures. Male Sprague Dawley rats were injected with PBS or G-CSF during (1) extinction or (2) abstinence from cocaine self-administration, and drug seeking behavior was measured. Quantitative assessment of changes in the proteomic landscape in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were performed via data-independent acquisition (DIA) mass spectrometry analysis. Administration of G-CSF during extinction accelerated the rate of extinction, and administration during abstinence attenuated cue-induced cocaine-seeking. Analysis of global protein expression demonstrated that G-CSF regulated proteins primarily in mPFC that are critical to glutamate signaling and synapse maintenance. Taken together, these findings support G-CSF as a viable translational research target with the potential to reduce drug craving or seeking behaviors. Importantly, recombinant G-CSF exists as an FDA-approved medication which may facilitate rapid clinical translation. Additionally, using cutting-edge multiregion discovery proteomics analyses, these studies identify a novel mechanism underlying G-CSF effects on behavioral plasticity.SIGNIFICANCE STATEMENT Pharmacological treatments for psychostimulant use disorder are desperately needed, especially given the disease's chronic, relapsing nature. However, there are currently no Food and Drug Administration (FDA)-approved pharmacotherapies. Emerging evidence suggests that targeting the immune system may be a viable translational research strategy; preclinical studies have found that the neuroactive cytokine granulocyte-colony stimulating factor (G-CSF) alters cocaine reward and reinforcement and can enhance cognitive flexibility. Given this basis of evidence we studied the effects of G-CSF treatment on extinction and reinstatement of cocaine seeking. We find that administration of G-CSF accelerates extinction and reduces cue-induced drug seeking after cocaine self-administration. In addition, G-CSF leads to downregulation of synaptic glutamatergic proteins in medial prefrontal cortex (mPFC), suggesting that G-CSF influences drug seeking via glutamatergic mechanisms.

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. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

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 O-phospho-l-tyrosine (pY). However, recent improvements in sample preparation, coupled with advances in instrument sensitivity and accessibility, have led to results that unequivocally disproved this assumption. Identification of hundreds of pY-peptides/proteins, followed by validation using genomic, molecular, and biochemical approaches, implies previously unappreciated roles for this "animal PTM" in plants. Herein, we review extant results from studies of pY in plants and propose a strategy for preparation and analysis of pY-peptides that will allow a depth of coverage of the plant pY-proteome comparable to that achieved in mammalian systems.

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. Using peptide information files exported from mass spectrometry search engines or quantitative tools as input, the peptide sequences are aligned to an internal protein database such as UniProtKB. Each identified peptide ion including those with PTMs is then visualized along the parent protein in the Browser. A unique property of ProteomicsBrowser is the ability to combine overlapping peptides in different ways to focus analysis of sequence coverage, charge state or PTMs. ProteomicsBrowser includes other useful functions, such as a data filtering tool and basic statistical analyses to qualify quantitative data. AVAILABILITY AND IMPLEMENTATION: ProteomicsBrowser is implemented in Java8 and is available at https://medicine.yale.edu/keck/nida/proteomicsbrowser.aspx and https://github.com/peng-gang/ProteomicsBrowser. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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. The committed step of de novo fatty acid synthesis in the plastids of most plants is catalyzed by the multienzyme, heteromeric acetyl-CoA carboxylase (hetACCase). This complex is composed of four catalytic subunits and a recently discovered regulatory subunit resembling the biotin carboxyl carrier protein but lacking the biotinylation motif necessary for activity. To better understand this novel form of regulation, a targeted tandem mass-spectrometry-based assay was developed to absolutely quantify all subunits to the Arabidopsis thaliana hetACCase. After validation against pure, recombinant protein, this multiplexed assay was used to quantify hetACCase subunits in siliques in various stages of development. Quantitation provided a developmental profile of hetACCase and BADC protein expression that supports a recently proposed regulatory mechanism for hetACCase and demonstrates a promising application of targeted mass spectrometry for in vivo analysis of protein complexes.

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. High-throughput liquid chromatography tandem mass spectrometry proteomics (LC-MS/MS) relies on enzymatic digestion and has lower coverage and sensitivity than transcriptomic profiling methods such as RNA-seq. Digestion results in predictable fragmentation of a protein, which can limit the generation of peptides capable of distinguishing between isoforms. Here we exploit transcript-level expression from RNA-seq to set prior likelihoods and enable protein isoform abundances to be directly estimated from LC-MS/MS, an approach derived from the principle that most genes appear to be expressed as a single dominant isoform in a given cell type or tissue. Through this deep integration of RNA-seq and LC-MS/MS data from the same sample, we show that a principal isoform can be identified in >80% of gene products in homogeneous HEK293 cell culture and >70% of proteins detected in complex human brain tissue. We demonstrate that the incorporation of translatome data from ribosome profiling further refines this process. Defining isoforms in experiments with matched RNA-seq/translatome and proteomic data increases the functional relevance of such data sets and will further broaden our understanding of multilevel control of gene expression.

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. Lignin content and composition are the most important factors associated with biomass recalcitrance to convert cell-wall polysaccharides into fermentable sugars. Thus it is important to understand the metabolic relationship between nitrogen fertilization and lignin in this feedstock. In this study, a large-scale proteomics approach based on GeLC-MS/MS was employed to identify and relatively quantify proteins differently accumulated in two contrasting genotypes for lignin composition after excessive nitrogen fertilization. From the ∼1000 nonredundant proteins identified, 28 and 177 were differentially accumulated in response to nitrogen from IACSP04-065 and IACSP04-627 lines, respectively. These proteins were associated with several functional categories, including carbon metabolism, amino acid metabolism, protein turnover, and oxidative stress. Although nitrogen fertilization has not changed lignin content, phenolic acids and lignin composition were changed in both species but not in the same way. Sucrose and reducing sugars increased in plants of the genotype IACSP04-065 receiving nitrogen.

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. Using an MS-based quantitative assay we demonstrate phosphorylation of 14-3-3 χ and ε at a total of seven sites, one of which is an in vivo site discovered in Arabidopsis. CPK autophosphorylation was also comprehensively monitored by MS and revealed a total of 45 sites among the six CPKs analysed, most of which were located within the N-terminal variable and catalytic domains. Among these CPK autophosphorylation sites was Tyr463 within the calcium-binding EF-hand domain of CPK28. Of all CPKs assayed, CPK28, which contained an autophosphorylation site (Ser43) within a canonical 14-3-3-binding motif, showed the highest activity against 14-3-3 proteins. Phosphomimetic mutagenesis of Ser72 to aspartate on 14-3-3χ, which is adjacent to the 14-3-3-binding cleft and conserved among all 14-3-3 isoforms, prevented 14-3-3-mediated inhibition of phosphorylated nitrate reductase.

Decoding Arabidopsis thaliana 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 Arabidopsis thaliana, 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. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

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. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based (LC-MS/MS) quantitative proteomics, along with a data-independent acquisition (DIA) analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveals cocaine and morphine differentially alters diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome.

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. Experimental groups included two mouse strains (C3H/HeJ and C57BL/6J) at baseline, a sub-chronic, rewarding regimen of nicotine in C3H/HeJ mice, and chronic nicotine administration and withdrawal in C57BL/6J mice. Isobaric labeling with a TMT 10-plex system, sample fractionation, and tandem mass spectrometry were used to quantify changes in protein abundance. In C3H/HeJ mice, similar numbers of proteins were differentially regulated between sexes at baseline compared with within each sex after sub-chronic nicotine administration. In C57BL/6J mice, there were significantly greater numbers of proteins differentially regulated between sexes at baseline compared with within each sex after chronic nicotine administration and withdrawal. Despite differences by sex, strain, and nicotine exposure parameters, glial fibrillary acidic protein (GFAP) and dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32, Ppp1r1b) were repeatedly identified as significantly altered proteins, especially in the VTA. Further, network analyses showed sex- and nicotine-dependent regulation of a number of signaling pathways, including dopaminergic signaling. Sub-chronic nicotine exposure in female mice increased proteins related to dopaminergic signaling in the NAc shell but decreased them in the VTA, whereas the opposite pattern was observed in male mice. In contrast, dopaminergic signaling pathways were similarly upregulated in both male and female VTA after chronic nicotine and withdrawal. Overall, this study identifies significant sex differences in the proteome of the mesolimbic system, at baseline and after nicotine reward or withdrawal, which may help explain differential trajectories and susceptibility to nicotine addiction in males and females.

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. This methodology is species independent; thus, while initially developed in a rodent model, we utilized this technique to analyze 36 phosphorylation sites on rhesus monkey tau from the prefrontal cortex (PFC), a region vulnerable to AD-linked degeneration. Data are available via ProteomeXchange with identifier PXD027971. We identified novel, age-related changes in tau phosphorylation in the rhesus monkey PFC and analyzed patterns of phosphorylation change across domains of the protein. We confirmed a significant increase and positive correlation with age of phosphorylated serine 235 tau and phosphorylated serine 396 tau levels in an expanded cohort of 14 monkeys. Histology showed robust labeling for tau phosphorylated at these sites in vulnerable layer III pyramidal cells in the PFC. The results presented in this study suggest an important role of the natural aging process in tau phosphorylation in rhesus monkey.

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. Here, we show that sex is a critical biological variable that defines drug-induced plasticity in the nucleus accumbens (NAc). Using quantitative mass spectrometry, we assessed the protein expression patterns induced by cocaine self-administration and demonstrated unique molecular profiles between males and females. We show that 1. Cocaine self-administration induces non-overlapping protein expression patterns in significantly regulated proteins in males and females and 2. Critically, cocaine-induced protein regulation differentially interacts with sex to eliminate basal sexual dimorphisms in the proteome. Finally, eliminating these baseline differences in the proteome is concomitant with the elimination of sex differences in behavior for non-drug rewards. Together, these data suggest that cocaine administration is capable of rewriting basal proteomic function and reward-associated behaviors.

Paper-based microfluidic devices for analysis of clinically relevant analytes present in urine and saliva.

We report the use of paper-based microfluidic devices fabricated from a novel polymer blend for the monitoring of urinary ketones, glucose, and salivary nitrite. Paper-based devices were fabricated via photolithography in less than 3 min and were immediately ready for use for these diagnostically relevant assays. Patterned channels on filter paper as small as 90 microm wide with barriers as narrow as 250 microm could be reliably patterned to permit and block fluid wicking, respectively. Colorimetric assays for ketones and nitrite were adapted from the dipstick format to this paper microfluidic chip for the quantification of acetoacetate in artificial urine, as well as nitrite in artificial saliva. Glucose assays were based on those previously demonstrated (Martinez et al., Angew Chem Int Ed 8:1318-1320, 1; Martinez et al., Anal Chem 10:3699-3707, 2; Martinez et al., Proc Nat Acad Sci USA 50:19606-19611, 3; Lu et al., Electrophoresis 9:1497-1500, 4; Abe et al., Anal Chem 18:6928-6934, 5). Reagents were spotted on the detection pad of the paper device and allowed to dry prior to spotting of samples. The ketone test was a two-step reaction requiring a derivitization step between the sample spotting pad and the detection pad, thus for the first time, confirming the ability of these paper devices to perform online multi-step chemical reactions. Following the spotting of the reagents and sample solution onto the paper device and subsequent drying, color images of the paper chips were recorded using a flatbed scanner, and images were converted to CMYK format in Adobe Photoshop CS4 where the intensity of the color change was quantified using the same software. The limit of detection (LOD) for acetoacetate in artificial urine was 0.5 mM, while the LOD for salivary nitrite was 5 microM, placing both of these analytes within the clinically relevant range for these assays. Calibration curves for urinary ketone (5 to 16 mM) and salivary nitrite (5 to 2,000 microM) were generated. The time of device fabrication to the time of test results was about 25 min.

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. The striatum contains two major cell types, D1 and D2 type dopaminoceptive medium spiny neurons (MSNs), whose cell bodies and processes are intermingled throughout this region. Since little is known about the proteomes of these two neuronal cell populations, we have begun to address this challenge by using fluorescence-activated nuclear sorting (FANS) to isolate nuclei-containing fractions from striatum from D1 and D2 "Translating Ribosome Affinity Purification" (TRAP) mice. This approach enabled us to devise and implement a robust and reproducible workflow for preparing samples from specific MSN cell types for mass spectrometry analyses. These analyses quantified at least 685 proteins in each of four biological replicates of 50 K sorted nuclei from two D1 mice/replicate and from each of four biological replicates of 50 K sorted nuclei from two D2 mice/replicate. Proteome analyses identified 87 proteins that were differentially expressed in D1 versus D2 MSN nuclei and principal component analysis (PCA) of these proteins separated the 8 biological replicates into specific cell types. Central network analysis of the 87 differentially expressed proteins identified Hnrnpd and Hnmpa2b1 in D1 and Cct2 and Cct7 in D2 as potential central interactors. This workflow can now be used to improve our understanding of many neurological diseases including characterizing the short and long-term impact of drugs of abuse on the proteomes of these two dopaminoceptive neuronal populations.

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. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue.

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 1.0101, Len c 1.0102, and lipid transfer proteins (LTPs), indicating qualitative and quantitative variations in allergen proteins in lentil cultivars. In this report, for the first-time, the amino acid sequence of LTPs in lentil has been confirmed by high resolution mass spectrometry. In addition, ideal peptides of Len c 1.0101, Len c 1.0102, and LTPs allergens were further determined with multiple reaction monitoring (MRM) analysis. Therefore, this data could provide a great resource for further development of targeted, proteomics-based assays for quantification of lentil allergens.

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. Despite these challenges, methods to improve proteomic yield and increase resolution continue to develop at a rapid rate. In this review, we highlight the importance of cell-type-specific proteomics in neuroscience and the technical difficulties associated. Furthermore, current progress and technological advancements in cell-type-specific proteomics research are discussed with an emphasis in neuroscience.