Biology subjects

Phinney, B. S.

Publications and source records attributed to Phinney, B. S..

8 recordsLinked to original sources

bioRxiv2025

Acute in vivo proximity labeling for membrane targeted proteomics in neuronal circuits

MOTIVATIONDespite the growing use of proximity labeling tools, there are limited techniques available to identify subcellularly localized proteins within targeted neuronal circuits on acute timescales in vivo. We engineered membrane targeted versions of the proximity labeling enzyme TurboID and validated their use for proteomic discovery in the mouse brain. We optimized an in vivo protocol to identify proteins in cell bodies and long range neuronal projections, and proteins differentially detected during acute drug labeling windows. SUMMARYA major goal within molecular systems neuroscience is to bridge the study of neuronal circuit function with changes in protein expression and localization in awake behaving animals. However, there are limited tools for capturing changes in subcellularly-defined proteomes within neuronal circuits during activity-gated timescales in vivo. Here, we engineered targeted versions of the proximity labeling enzyme TurboID, to tag proteins at the neuronal membrane during a user-delivered biotin injection. We optimized a labeling strategy that enables a one-to-two-hour labeling window and tagged proteins in medial prefrontal cortex (mPFC) cell bodies and corresponding axons in a downstream projection. We performed proteomics to identify proteins enriched in mPFC cell bodies and terminals, and upregulated in mPFC cell bodies following an acute cocaine injection. These advancements enable the detection of proteins at the subcellular level within short labeling windows, allowing identification of stimulus-specific proteomes in behaving mice.

neuroscience↗

bioRxiv2025

Development and validation of a streamlined workflow for proteomic analysis of proteins and post-translational modifications from dried blood

It is increasingly recognized that the omic analysis of whole blood has applications for precision medicine and disease phenotyping. Despite this realization, whole blood is generally viewed as a challenging analytical matrix in comparison to plasma or serum. Moreover, proteomic analyses of whole blood proteomics have almost exclusively focused on (non)targeted analyses of protein abundances and much less on post-translational modifications (PTMs). Here, we developed a streamlined workflow for processing twenty microliters of venous blood collected by volumetric absorptive microsampling that incorporates serial trypsinization, N-glycopeptide and phosphopeptide enrichment and avoids laborious sample dry-down or cleanup steps. Up to 10,000 analytes (reported as protein groups, glycopeptidoforms and phosphosites) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in approximately 2 h of MS acquisition time. Using these methods, we explored the stability of "dried" and "wet" blood proteomes, as well as effects of ex vivo inflammatory stimulus or phosphatase inhibition. Multi-omics factor analysis enabled facile identification of analytes that contributed to inter-individual variability of the blood proteomes, including N-glycopeptides that distinguish immunoglobulin heavy constant alpha 2 allotypes. Collectively, our results help to establish feasibility and best practices for the integrated MS-based quantification of proteins and PTMs from dried blood.

biochemistry↗

bioRxiv2024

Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function

ATG5 is one of the core autophagy proteins with additional functions such as noncanonical membrane atg8ylation, which among a growing number of biological outputs includes control of tuberculosis in animal models. Here we show that ATG5 associates with retromers core components VPS26, VPS29 and VPS35 and modulates retromer function. Knockout of ATG5 blocked trafficking of a key glucose transporter sorted by the retromer, GLUT1, to the plasma membrane. Knockouts of other genes essential for membrane atg8ylation, of which ATG5 is a component, affected GLUT1 sorting, indicating that membrane atg8ylation as a process affects retromer function and endosomal sorting. The contribution of membrane atg8ylation to retromer function in GLUT1 sorting was independent of canonical autophagy. These findings expand the scope of membrane atg8ylation to specific sorting processes in the cell dependent on the retromer and its known interactors.

cell biology↗

bioRxiv2024

The balance between antiviral and antibacterial responses during M. Tuberculosis infection is regulated by the ubiquitin ligase CBL

As a first line of host defense, macrophages must be able to effectively sense and respond to diverse types of pathogens, and while a particular type of immune response may be beneficial in some circumstances, it can be detrimental in others. Upon infecting a macrophage, M. tuberculosis (Mtb) induces proinflammatory cytokines that activate antibacterial responses. Surprisingly, Mtb also triggers antiviral responses that actually hinder the ability of macrophages to control Mtb infection. The ubiquitin ligase CBL suppresses these antiviral responses and shifts macrophages toward a more antibacterial state during Mtb infection, however, the mechanisms by which CBL regulates immune signaling are unknown. We found that CBL controls responses to multiple stimuli and broadly suppresses the expression of antiviral effector genes. We then used mass-spectrometry to investigate potential CBL substrates and identified over 46,000 ubiquitylated peptides in Mtb-infected macrophages, as well as roughly 400 peptides with CBL-dependent ubiquitylation. We then performed genetic interaction analysis of CBL and its putative substrates, and identified the Fas associated factor 2 (FAF2) adapter protein as a key signaling molecule protein downstream of CBL. Together, these analyses identify thousands of new ubiquitin-mediated signaling events during the innate immune response and reveal an important new regulatory hub in this response.

immunology↗

bioRxiv2024

Probe-dependent Proximity Profiling (ProPPr) Uncovers Similarities and Differences in Phospho-Tau-Associated Proteomes Between Tauopathies

Tauopathies represent a diverse group of neurodegenerative disorders characterized by the abnormal aggregation of the microtubule-associated protein tau. Despite extensive research, the precise mechanisms underlying the complexity of different types of tau pathology remain incompletely understood. Here we describe an approach for proteomic profiling of aggregate-associated proteomes on slides with formalin-fixed, paraffin-embedded (FFPE) tissue that utilizes proximity labelling upon high preservation of aggregate morphology, which permits the profiling of pathological aggregates regardless of their size. To comprehensively investigate the common and unique protein interactors associated with the variety of tau lesions present across different human tauopathies, Alzheimers disease (AD), corticobasal degeneration (CBD), Picks disease (PiD), and progressive supranuclear palsy (PSP), were selected to represent the major tauopathy diseases. Implementation of our widely applicable Probe-dependent Proximity Profiling (ProPPr) strategy, using the AT8 antibody, permitted identification and quantification of proteins associated with phospho-tau lesions in well-characterized human post-mortem tissue. The analysis revealed both common and disease-specific proteins associated with phospho-tau aggregates, highlighting potential targets for therapeutic intervention and biomarker development. Candidate validation through high-resolution co-immunofluorescence of distinct aggregates across disease and control cases, confirmed the association of retromer complex protein VPS35 with phospho-tau lesions across the studied tauopathies. Furthermore, we discovered disease-specific associations of proteins including ferritin light chain (FTL) and the neuropeptide precursor VGF within distinct pathological lesions. Notably, examination of FTL-positive microglia in CBD astrocytic plaques indicate a potential role for microglial involvement in the pathogenesis of these tau lesions. Our findings provide valuable insights into the proteomic landscape of tauopathies, shedding light on the molecular mechanisms underlying tau pathology. This first comprehensive characterization of tau-associated proteomes across different tauopathies enhances our understanding of disease heterogeneity and provides a resource for future functional investigation, as well as development of targeted therapies and diagnostic biomarkers.

neuroscience↗

bioRxiv2021

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of matching non-synonymous single nucleotide polymorphisms for the individual who originated the protein sample. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of genetically variant peptides increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data independent acquisition and a variant of parallel reaction monitoring on a Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring on an Agilent 6495 triple quadrupole system. In our hands, average genetically variant peptide detection from a selected 24 genetically variant peptide panel increased from 6.5 {+/-} 1.1 and 3.1 {+/-} 0.8 using data dependent and independent acquisition to 9.5 {+/-} 0.7 and 11.7 {+/-} 1.7 using parallel reaction monitoring and multiple reaction monitoring (p < 0.05). Parallel reaction monitoring resulted in a 1.3-fold increase in detection sensitivity, and multiple reaction monitoring resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For parallel reaction monitoring and multiple reaction monitoring, detected genetically variant peptides were validated by the inclusion of stable isotope labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping, and the establishment of validation methods for the evaluation of inferred genotypes. HighlightsO_LITest four mass spectrometry configurations to optimize detection of genetically variant peptides C_LIO_LITechnology transfer of proteomic genotyping assays C_LIO_LIImproved sensitivity results in higher level of forensic discrimination for human identification using multiple reaction monitoring C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/435505v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1001cfaorg.highwire.dtl.DTLVardef@6e8aa1org.highwire.dtl.DTLVardef@14f8076org.highwire.dtl.DTLVardef@7ad930_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

2019 Association of Biomolecular Resource Facilities Multi-Laboratory Data-Independent Acquisition Study

Despite the advantages of fewer missing values by collecting fragment ion data on all analytes in the sample, as well as the potential for deeper coverage, the adoption of data-independent acquisition (DIA) in core facility settings has been slow. The Association of Biomolecular Resource Facilities conducted a large interlaboratory study to evaluate DIA performance in laboratories with various instrumentation. Participants were supplied with generic methods and a uniform set of test samples. The resulting 49 DIA datasets act as benchmarks and have utility in education and tool development. The sample set consisted of a tryptic HeLa digest spiked with high or low levels of four exogenous proteins. Data are available in MassIVE MSV000086479. Additionally, we demonstrate how the data can be analysed by focusing on two datasets using different library approaches and show the utility of select summary statistics. These data can be used by DIA newcomers, software developers, or DIA experts evaluating performance with different platforms, acquisition settings and skill levels.

bioinformatics↗

bioRxiv2020

Deep learning neural network prediction method improves proteome profiling of vascular sap of grapevines during Pierce's disease development

Plant secretome studies have shown the importance of plant defense proteins in the vascular system against pathogens. Studies on Pierces disease of grapevines caused by the xylem-limited bacteria Xylella fastidiosa (Xf) have detected proteins and pathways associated to its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to technical and technological challenges. Deep learning neural network prediction methods can provide powerful tools for improving proteome profiling by data-independent acquisition (DIA). We aimed to explore the potential of this strategy by combining it with in silico spectral library prediction tool, Prosit, to analyze the proteome of vascular leaf sap of grapevines with Pierces disease. The results demonstrate that the combination of DIA and Prosit increased the total number of identified proteins from 145 to 360 for grapevines and 18 to 90 for Xf. The new proteins increased the range of molecular weight, assisted on the identification of more exclusive peptides per protein, and increased the identification of low abundance proteins. These increases allowed the identification of new functional pathways associated with cellular responses to oxidative stress to be further investigated.

genomics↗