Biology subjects

Frey, B. L.

Publications and source records attributed to Frey, B. L..

3 recordsLinked to original sources

bioRxiv2025

Chemoselective Characterization of New Extracellular Matrix Deposition in Bioengineered Tumor Tissues

The extracellular matrix (ECM), present in nearly all tissues, provides extensive support to resident cells through structural, biomechanical, and biochemical means, and in return the ECM undergoes constant remodeling from interacting cells to adapt to the evolving tissue states. Bioengineered 3D tissues, commonly known as cell-ECM composites, are robust model systems to recapitulate and investigate native pathophysiology. Key to this engineered morphogenesis process are the intricate cell-ECM interactions reflected by how cells respond to and thereby modulate their surrounding microenvironments through their ongoing ECM secretome. However, investigating ECM-regulated new ECM production has been challenging due to the proteomic background from the pre-existing biomaterial ECM. To address this hindrance, here we present a chemoselective strategy to label, enrich, and characterize newly synthesized ECM (newsECM) proteins produced by resident cells, allowing distinction from the pre-existing ECM background. Applying our analytical pipeline to bioengineered tumor tissues, either built upon decellularized ECM (dECM-tumors) or as ECM-free tumor spheroids (tumoroids), we observed distinct ECM synthesis patterns that were linked to their extracellular environments. Tumor cells responded to the dECM presence with elevated ECM remodeling activities, mediated by augmented digestion of pre-existing ECM coupled with upregulated synthesis of tumor-associated ECM. Our findings highlight the sensitivity of newsECM profiling to capture remodeling events that are otherwise under-represented by bulk proteomics and underscore the significance of dECM support for enabling native-like tumor cell behaviors. We anticipate the described newsECM analytical pipeline to be broadly applicable to other tissue-engineered systems to probe ECM-regulated ECM synthesis and remodeling, both fundamental aspects of cell-ECM crosstalk in engineered tissue morphogenesis.

bioengineering↗

bioRxiv2024

Discovery of dehydroamino acids and their crosslinks in Tau and other aggregating proteins of Alzheimers disease

BackgroundAlzheimers disease (AD) is characterized by accumulation of two types of protein aggregates, senile plaques and neurofibrillary tangles, which strongly contribute to the pathogenesis of the disease. Senile plaques consist primarily of aggregated amyloid-{beta}, while neurofibrillary tangles form via aggregation of the protein Tau, as well as other microtubule-associated proteins such as CRMP2. Posttranslational modifications have been hypothesized to contribute to the initial aggregation events that lead to SPs and NFTs. Dehydroamino acids (DHAAs) are posttranslational modifications rarely observed in humans and have not previously been reported in AD. DHAAs arise from the eliminylation of serine, threonine, or cysteine, yielding a double bond with distinct molecular geometry and reactivity. Their geometry can produce secondary structure rearrangements, such as those seen in senile plaque and neurofibrillary tangle formation, while their reactivity can cause intramolecular or intermolecular (protein-protein) crosslinking. We hypothesized that this modification might be present in protein aggregation-associated neurodegenerative disorders like AD. MethodsWe performed mass spectrometry-based bottom-up proteomics on the sarkosyl-insoluble (protein aggregate-enriched) material from ten AD brains and three age-matched controls. Identifications of DHAA-mediated crosslinked peptides were validated using both an isotopic labeling strategy and spike-in experiments employing synthetic crosslinked peptide standards. Similar findings were obtained in searches of publicly available proteomic datasets from AD and control brains. ResultsWe identified 412 sites of DHAA modification in 184 proteins, with the highest prevalence in the neurofibrillary tangle-forming proteins Tau and CRMP2. Comparison with results of previous protein aggregate interactomics studies show that proteins containing the DHAA modification are more highly associated with protein aggregates than are proteins containing any other individual posttranslational modification. We further observed 11 protein crosslinks arising from DHAAs, including three from the Tau protein. Label free quantification showed that Tau crosslinks are an order of magnitude more prevalent in AD samples than in age-matched controls. ConclusionsDehydroamino acids and their derivatives are prevalent modifications in the Alzheimers disease brain proteome. These modifications give rise to protein crosslinks which may contribute to protein aggregation processes.

biochemistry↗

bioRxiv2020

Spritz: A Proteogenomic Database Engine

Proteoforms are the workhorses of the cell, and subtle differences between their amino acid sequence or post-translational modifications (PTMs) can change their biological function. To most effectively identify and quantify proteoforms in genetically diverse samples by mass spectrometry (MS), it is advantageous to search the MS data against a sample-specific protein database that is tailored to the sample being analyzed, in that it contains the correct amino acid sequences and relevant PTMs for that sample. To this end, we have developed Spritz (https://smith-chem-wisc.github.io/Spritz/), an open-source software tool for generating protein databases annotated with sequence variations and PTMs. We provide a simple graphical user interface (GUI) for Windows and scripts that can be run on any operating system. Spritz automatically sets up and executes approximately 20 tools, which enable construction of a proteogenomic database from only raw RNA sequencing data. Sequence variations that are discovered in RNA sequencing data upon comparison to the Ensembl reference genome are annotated on proteins in these databases, and PTM annotations are transferred from UniProt. Modifications can also be discovered and added to the database using bottom-up mass spectrometry data and global PTM discovery in MetaMorpheus. We demonstrate that such sample-specific databases allow the identification of variant peptides, modified variant peptides, and variant proteoforms by searching bottom-up and top-down proteomic data from the Jurkat human T lymphocyte cell line and demonstrate the identification of phosphorylated variant sites with phosphoproteomic data from the U2OS human osteosarcoma cell line.

bioinformatics↗