Biology subjects

Barshop, W. D.

Publications and source records attributed to Barshop, W. D..

5 recordsLinked to original sources

bioRxiv2024

Function and interactions of a protein bridge between the inner membrane complex and subpellicular microtubules in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that utilizes peripheral membrane and cytoskeletal structures for critical functions such as host cell invasion, replication, and maintaining cellular morphology. These structures include the inner membrane complex (IMC) as well as the underlying longitudinal subpellicular microtubules (SPMTs) that provide support for the IMC and give the parasite its distinctive crescent shape. While the IMC and SPMTs have been studied on their own, the mechanisms linking these adjacent structures remain largely unknown. This study identifies a T. gondii protein named IMT1 that localizes to the maternal IMC and SPMTs and thus appears to tether the IMC to the microtubules. We disrupt the IMT1 gene to assess function and then use deletion analyses and mutagenesis to reveal regions of the protein that are necessary for binding to the IMC cytoskeleton or SPMTs. Using proximity labelling with IMT1 as bait, we identify a series of candidate interactors in the IMC or SPMTs. Exploration of two of these candidates reveals that IMT1 regulates the levels of the microtubule associated protein TLAP2 and binds directly to the cytoskeletal IMC protein IMC1. Taken together, these interactions unveil the specific interactions linking two key cytoskeletal structures of the parasite and provides new insight into the organization of the structural backbone of T. gondii.

microbiology↗

bioRxiv2023

Real-time spectral library matching for sample multiplexed quantitative proteomics.

Sample multiplexed quantitative proteomics has proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor co-isolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Towards this end, we sought to develop and implement a new real-time library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time as traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy in sample multiplexed analyses.

cell biology↗

bioRxiv2022

Dynamic Data Independent Acquisition Mass Spectrometry with Real-Time Retrospective Alignment

We report a data independent acquisition (DIA) strategy that dynamically adjusts the tandem mass spectrometry (MS/MS) windows during the chromatographic separation. The method focuses MS/MS acquisition on the most relevant mass range at each point in time - improving the quantitative sensitivity by increasing the time spent on each DIA window. We demonstrate an improved lower limit of quantification, on average, without sacrificing the number of peptides detected.

bioinformatics↗

bioRxiv2021

Novel Real-Time Library Search driven data acquisition strategy for identification and characterization of metabolites.

Identification and structural characterization of novel metabolites in drug discovery or metabolomics experiments is one of the most challenging tasks. Multi-level fragmentation (MSn) based approaches combined with various dissociation modes are frequently utilized for facilitating structure assignment of the unknown compounds. As each of the MS precursors undergoes MSn, the instrument cycle time can limit the total number of precursors analyzed in a single run for complex samples. This necessitates splitting data acquisition into several LC/MS analyses where the results obtained in one acquisition inform the experimental design for the successive experiment. Here we present a new LC/MS data acquisition strategy, termed Met-IQ, where the decision to perform an MSn acquisition is automatically made in real time based on the similarity between an acquired experimental MS2 spectrum and a spectrum in a reference spectral library. Each MS2 spectrum is searched in real time against the spectra for the known compounds of interest. If a similarity to a spectrum in the library is found, the instrument performs a decision-dependent event, such as an MS3 scan. Compared to an intensity-based, data-dependent MSn experiment, only a selective number of MS3 are triggered using Met-IQ, increasing the overall MS2 instrument sampling rate. We applied this strategy to an Amprenavir sample incubated with human liver microsomes. The number of MS2 scan events increased nearly 3.5-fold compared to the standard data dependent experiment where MS3 was triggered for each precursor ion, resulting in identification and structural characterization of 55% more unique metabolites. Furthermore, the MS3 precursor fragments were selected intelligently, focusing on higher mass fragments of sufficient intensity to maximize acquisition of MS3 data relevant for structure assignment. The described Met-IQ strategy is not limited to metabolism experiments, and can be applied to analytical samples where the detection of unknown compounds structurally related to a known compound(s) is sought.

pharmacology and toxicology↗

bioRxiv2020

Adenovirus E1A Binding to DCAF10 Targets Degradation of AAA+ ATPases Required for Quaternary Assembly of Multiprotein Machines and Innate Immunity

Adenovirus E1A early proteins modify host cell physiology to optimize virus replication. The N-terminal half of small e1a interacts with RB-family proteins to de-repress dNTP and DNA synthesis, and with p300/CBP to inhibit host anti-viral innate immune responses. These e1a N-terminal interactions activate a strong, late host anti-viral response due to stabilization and activation of interferon response factor 3 (IRF3). However, the C-terminal half of e1a inhibits this through interactions with three host proteins with seemingly unrelated functions. Proteomic analysis showed that all three C-terminal interactions are required for e1a-association into an [~]1 MDa multi-protein complex with scaffold subunits of a CRL4 E3 ubiquitin ligase and DCAF10, a presumed specificity subunit. This e1a-DCAF10-CRL4 prevents IRF3 stabilization indirectly by directing degradation of the essential AAA+ ATPases RUVBL1/2, subunits of several HSP90 co-chaperones required for quaternary assembly of cellular protein machines required for anti-viral defenses and responses to genotoxic and metabolic stress.

microbiology↗