Biology subjects

Merrihew, G. E.

Publications and source records attributed to Merrihew, G. E..

14 recordsLinked to original sources

bioRxiv2026

Circulating extracellular vesicles in plasma carry accessible molecular signatures of aging in mice

Cells release membrane-bound extracellular vesicles into the bloodstream laden with proteins that may reflect their physiological state. How this circulating EV proteome changes across life remains poorly understood. Identifying molecular signatures of aging in accessible biofluids could facilitate earlier intervention and monitoring of age-related disease. Many circulating aging proteome studies rely on affinity-based platforms which suffer from poor cross-species translation, ambiguous signal attribution, and inconsistent agreement between platforms. Here, we present a characterization of the aging plasma EV proteome from a cross-sectional cohort of 86 male and female C57BL/6J mice (5-31 months). We leveraged a species-agnostic EV enrichment (Mag-Net) and mass spectrometry to detect 2,575 protein groups from 15,969 peptides. Protein abundance heterogeneity increased with age and the abundance of 272 proteins were significantly correlated with chronological age including established senescence and frailty markers. Proteins increasing with age were enriched in genome maintenance pathways, while those decreasing were associated with the extracellular matrix organization and lipid metabolism. Notably, several of the strongest age-increased proteins converged on Alzheimers and Parkinsons disease pathology. We observed sexual divergence in the aging EV proteome not previously characterized at this resolution. A proteomic clock built from this data accurately predicts chronological age, and peptide-level analysis reveals aging signals invisible at protein-level. These findings demonstrate that EV-enriched plasma proteomics can identify known aging markers, reveal novel sex-specific age-related changes, and generate predictive models of chronological age. This study provides a species-agnostic foundation for proteomic clocks that complement epigenetic approaches to monitor aging and evaluate healthspan.

molecular biology↗

bioRxiv2026

A quantitative proteomics dataset for assessment and prediction of low dose X-ray radiation exposure in mice.

Ionizing radiation induces molecular responses that may be used to estimate exposure when physical dosimeters are unavailable. Here we present two large-scale proteomics datasets generated from mouse dorsal skin punch samples collected following controlled X-ray exposures spanning multiple doses, dose rates, and post-exposure time points. Experiment 1 comprised 96 samples (including 16 reference samples) collected 6 days after exposure to 0-75 cGy delivered at either 30 or 300 cGy/min. Experiment 2 comprised 936 samples (including 236 reference samples) exposed to 0-100 cGy at either 3 or 28 cGy/min dose rates and harvested between 7 and 150 days post-exposure. All samples were processed using a standardized workflow involving automated bead-based digestion and data-independent acquisition mass spectrometry. The datasets include multiple pooled reference sample types, process controls, and system suitability standards ensuring high quality data. All data presented are available via ProteomeXchange at several levels of processing, from raw files through normalized peptide- and protein-level abundance matrices suitable for biomarker discovery and machine learning applications. This dataset will facilitate generation of new insights into the biological changes and molecular signatures resulting from X-ray exposure in mice and may also help inform future studies in humans.

biochemistry↗

bioRxiv2026

Is Protein Quantification and Physical Normalization Always Necessary in Proteomics?

Dogma suggests protein quantification is a pre-requisite to LC-MS/MS based proteomics studies. Such quantification allows a standardized ratio of sample to digestion enzyme and enables physical normalization of protein digest loaded onto the mass spectrometer for analysis. Most proteomics studies include these steps. However, there are significant costs in time, money and experimental complexity, associated with performing protein quantification and physical normalization for every sample, especially for larger studies. Proteomics data analysis pipelines typically include computational normalization strategies to compensate for unavoidable systematic biases. These strategies also have the potential to compensate for avoidable variation such as omitting sample amount normalization. Here we investigate the effects of either physically normalizing the amount of protein for each individual sample or leaving it unnormalized. Our results show the relationship between increased protein amount variation in sample input, and the variance of quantified relative abundances of peptides and proteins output after data analysis. The experiments presented here suggest that protein quantification and physical normalization steps can be omitted from some quantitative proteomic experiments without incurring an unacceptable increase in measurement variability after computational normalization has been applied. This work will enable important time and cost saving optimizations to be made to many proteomics workflows.

biochemistry↗

bioRxiv2024

A second X chromosome improves cognition in aging male and female mice

Women show resilience to cognitive aging, in the absence of dementia, in many populations. To dissect sex differences, we utilized the FCG and XY*mouse models. Female gonads and sex chromosomes improved cognition in aging mice of both sexes. Further, presence of a second X in male and female mice conferred cognitive resilience while its absence in females blocked it. In the hippocampal proteome of aging female mice, the second X increased proteins involved in synaptogenesis signaling - a potential pathway to improved cognition.

neuroscience↗

bioRxiv2024

Development of highly multiplex targeted proteomics assays in biofluids using the Stellar mass spectrometer

The development of targeted assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to large scale clinical studies. Targeted assays are currently unable to scale to hundreds or thousands of targets. We demonstrate the generation of large-scale assays using a novel hybrid nominal mass instrument. The scale of these assays is achievable with the StellarTM mass spectrometer through the accommodation of shifting retention times by real-time alignment, while being sensitive and fast enough to handle many concurrent targets. Assays were constructed using precursor information from gas-phase fractionated (GPF) data-independent acquisition (DIA). We demonstrate the ability to schedule methods from an orbitrap and linear ion trap acquired GPF DIA library and compare the quantification of a matrix-matched calibration curve from orbitrap DIA and linear ion trap parallel reaction monitoring (PRM). Two applications of these proposed workflows are shown with a cerebrospinal fluid (CSF) neurodegenerative disease protein PRM assay and with a Mag-Net enriched plasma extracellular vesicle (EV) protein survey PRM assay.

biochemistry↗

bioRxiv2024

A framework for quality control in quantitative proteomics

A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow from planning to analysis. We share vignettes applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted methods, and we share examples where they are used on three instrument platforms to identify severe system failures and track function over months to years. Internal QCs incorporated at protein and peptide-level allow our team to assess sample preparation issues and to differentiate system failures from sample-specific issues. External QC samples prepared alongside our experimental samples are used to verify the consistency and quantitative potential of our results during batch correction and normalization before assessing biological phenotypes. We combine these controls with rapid analysis (Skyline), longitudinal QC metrics (AutoQC), and server-based data deposition (PanoramaWeb). We propose that this integrated approach to QC is a useful starting point for groups to facilitate rapid quality control assessment to ensure that valuable instrument time is used to collect the best quality data possible. Data are available on Panorama Public and on ProteomeXchange under the identifier PXD051318.

biochemistry↗

bioRxiv2024

Hippocampus Glutathione S Reductase Potentially Confers Genetic Resilience to Cognitive Decline in the of AD-BXD Mouse Population

Alzheimers disease (AD) is a prevalent and costly age-related dementia. Heritable factors account for 58-79% of variation in late-onset AD, but substantial variation remains in age-of- onset, disease severity, and whether those with high-risk genotypes acquire AD. To emulate the diversity of human populations, we utilized the AD-BXD mouse panel. This genetically diverse resource combines AD genotypes with multiple BXD strains to discover new genetic drivers of AD resilience. Comparing AD-BXD carriers to noncarrier littermates, we computed a novel quantitative metric for resilience to cognitive decline in the AD-BXDs. Our quantitative AD resilience trait was heritable and genetic mapping identified a locus on chr8 associated with resilience to AD mutations that resulted in amyloid brain pathology. Using a hippocampus proteomics dataset, we nominated the mitochondrial glutathione S reductase protein (GR or GSHR) as a resilience factor, finding that the DBA/2J genotype was associated with substantially higher GR abundance. By mapping protein QTLs (pQTLs), we identified synaptic organization and mitochondrial proteins coregulated in trans with a cis-pQTL for GR. We found four coexpression modules correlated with the quantitative resilience score in aged 5XFAD mice using paracliques, which were related to cell structure, protein folding, and postsynaptic densities. Finally, we found significant positive associations between human GSR transcript abundance in the brain and better outcomes on AD-related cognitive and pathology traits in the Religious Orders Study/Memory and Aging project (ROSMAP). Taken together, these data support a framework for resilience in which neuronal antioxidant pathway activity provides for stability of synapses within the hippocampus.

neuroscience↗

bioRxiv2023

Comparing peptide identifications by FAIMS versus quadrupole gas-phase fractionation

High-field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to liquid chromatography-mass spectrometry (LC-MS) has been shown to increase peptide and protein detections compared to LC-MS/MS alone. However, FAIMS has not been compared to other methods of gas-phase fractionation, such as quadrupole gas-phase fractionation, which could increase our understanding of the mechanisms of improvement. The goal of this work was to assess whether FAIMS improves peptide identifications because 1) gas-phase fractionation enables the analysis of less abundant signals by excluding more abundant precursors from filling the ion trap, 2) the use of FAIMS reduces co-isolation of peptides during the MS/MS process resulting in a reduction of chimeric spectra, or 3) a combination of both. To investigate these hypotheses, pooled human brain tissue samples were measured in triplicate using FAIMS gas-phase fractionation, quadrupole gas-phase fractionation, or no gas-phase fractionation on two Thermo Eclipse Tribrid Mass Spectrometers. On both instruments, our data confirmed prior observations that FAIMS increased the number of peptides identified. We further demonstrated that the main benefit of FAIMS is due to the reduced co-isolation of persistent peptide precursor ions, which results in a decrease in chimeric spectra.

biochemistry↗

bioRxiv2022

A Peptide-Centric Quantitative Proteomics Dataset for the Phenotypic Assessment of Alzheimer's Disease

Alzheimers disease (AD) is a looming public health disaster with limited interventions. Alzheimers is a complex disease that can present with or without causative mutations and can be accompanied by a range of age-related comorbidities. This diverse presentation makes it difficult to study molecular changes specific to AD. To better understand the molecular signatures of disease we constructed a unique human brain sample cohort inclusive of autosomal dominant AD dementia (ADD), sporadic ADD, and those without dementia but with high AD histopathologic burden, and cognitively normal individuals with no/minimal AD histopathologic burden. All samples are clinically well characterized, and brain tissue was preserved postmortem by rapid autopsy. Samples from four brain regions were processed and analyzed by data-independent acquisition LC-MS/MS. Here we present a high-quality quantitative dataset at the peptide and protein level for each brain region. Multiple internal and external control strategies were included in this experiment to ensure data quality. All data are deposited in the ProteomeXchange repositories and available from each step of our processing.

neuroscience↗

bioRxiv2022

Proteomics of resilience to Alzheimer's disease identifies brain regional soluble Aβ levels, actin filament processes, and response to injury

Resilience to Alzheimers disease (RAD) is an uncommon combination of high disease burden without dementia that may provide critical insights into limiting the clinical impact of this incurable disease. In this study, we used mass spectrometry-based proteomics to quantify regional protein differences that characterize RAD. Starting with over 700 brain donations, we identified 43 extensively annotated research participants who met stringent inclusion exclusion criteria and analyzed matched isocortical regions, hippocampus, and caudate nucleus. Differential expression analysis of 7,115 soluble proteins identified lower isocortical and hippocampal soluble A{beta} peptide levels as a significant feature of RAD. Protein co-expression analysis revealed a group of 181 densely-interacting proteins significantly associated with RAD that were enriched for actin filament-based process, cellular detoxification, and wound healing in isocortex and hippocampus. We further support our findings using data from 689 human isocortical samples from four independent external cohorts that were the closest approximations of our clinico-pathologic groups. The molecular basis of RAD, a widely replicated state in older adults for which there is no experimental model, likely holds important insights into therapeutic interventions for Alzheimers disease.

neuroscience↗

bioRxiv2021

Elamipretide (SS-31) Treatment Attenuates Age-Associated Post-Translational Modifications of Heart Proteins

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8-weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.

cell biology↗

bioRxiv2021

Spontaneous Isomerization of Asp387 in Tau is Diagnostic for Alzheimer's Disease: An Endogenous Indicator of Reduced Autophagic Flux

Amino acid isomerization is a spontaneous chemical modification potentially related to the underlying causes of Alzheimers disease (AD). We demonstrate that data-independent acquisition mass spectrometry can be used to characterize isomerization in complex protein mixtures. Examination of a large cohort of brain tissue samples revealed a striking relationship between isomerization of tau and AD status. Surprisingly, isomerization was found to be more abundant in both autosomal dominant and sporadic AD samples relative to controls. We hypothesize that lower autophagic flux in AD brains accounts for these results. Additional data, including quantitative analysis of proteins related to autophagy, strongly support this hypothesis. For example, isomerization of tau is positively correlated with levels of p62, a recognized indicator of autophagic inhibition. In sum, the data suggest strong ties between isomerization and autophagic flux, which may therefore represent a promising target for future investigations into the therapy and prevention of AD.

neuroscience↗

bioRxiv2020

Proteomics support the threespine stickleback egg coat as a protective oocyte envelope

After the end of the last ice age, ancestrally marine threespine stickleback fish (Gasterosteus aculeatus) have undergone an adaptive radiation into freshwater environments throughout the Northern Hemisphere, creating an excellent model system for studying molecular adaptation and speciation. Stickleback populations are reproductively isolated to varying degrees, despite the fact that they can be crossed in the lab to produce viable offspring. Ecological and behavioral factors have been suggested to underlie incipient stickleback speciation. However, reproductive proteins represent a previously unexplored driver of speciation. As mediators of gamete recognition during fertilization, reproductive proteins both create and maintain species boundaries. Gamete recognition proteins are also frequently found to be rapidly evolving, and their divergence may culminate in reproductive isolation and ultimately speciation. As an initial investigation into the contribution of reproductive proteins to stickleback reproductive isolation, we characterized the egg coat proteome of threespine stickleback eggs. In agreement with other teleosts, we find that stickleback egg coats are comprised of homologs to the zona pellucida (ZP) proteins ZP1 and ZP3. We explore aspects of stickleback ZP protein biology, including glycosylation, disulfide bonding, and sites of synthesis, and find many substantial differences compared to their mammalian homologs. Furthermore, molecular evolutionary analyses indicate that ZP3, but not ZP1, has experienced positive Darwinian selection across teleost fish. Taken together, these changes to stickleback ZP protein architecture suggest that the egg coats of stickleback fish, and perhaps fish more generally, have evolved to fulfill a more protective functional role than their mammalian counterparts. Data are available via ProteomeXchange with identifiers PXD017488 and PXD017489.

molecular biology↗

bioRxiv2018

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis.

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classesof molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsysamples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolateEVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C. elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on ascale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system.

cell biology↗