Biology subjects

MacCoss, M. J.

Publications and source records attributed to MacCoss, M. J..

69 records · Page 4Linked to original sources

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↗

bioRxiv2020

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

physiology↗

bioRxiv2019

A simple mass-action model predicts protein timecourses from mRNA trajectories during a dynamic response in two strains of S. cerevisiae

Although mRNA is a necessary precursor to protein, several studies have argued that the relationship between mRNA and protein levels is often weak. This claim undermines the functional relevance of conclusions based on quantitative analyses of mRNA levels, which are ubiquitous in modern biology from the single gene to the whole genome scale. Furthermore, if post-translational processes vary between strains and species, then comparative studies based on mRNA alone would miss an important driver of diversity. However, gene expression is dynamic, and most studies examining relationship between mRNA and protein levels at the genome scale have analyzed single timepoints. We measure yeast gene expression after pheromone exposure and show that, for most genes, protein timecourses can be predicted from mRNA timecourses through a simple, gene-specific, generative model. By comparing model parameters and predictions between strains, we find that while mRNA variation often leads to protein differences, evolution also manipulates protein-specific processes to amplify or buffer transcriptional regulation.

genomics↗

bioRxiv2019

Matrix-matched calibration curves for assessing analytical figures of merit in quantitative proteomics

Mass spectrometry is a powerful tool for quantifying protein abundance in complex samples. Advances in sample preparation and the development of data independent acquisition (DIA) mass spectrometry approaches have increased the number of peptides and proteins measured per sample. Here we present a series of experiments demonstrating how to assess whether a peptide measurement is quantitative by mass spectrometry. Our results demonstrate that increasing the number of detected peptides in a proteomics experiment does not necessarily result in increased numbers of peptides that can be measured quantitatively.

biochemistry↗

bioRxiv2019

Avant-garde: An automated data-driven DIA data curation tool.

Data-Independent Acquisition (DIA) is a technique that promises to comprehensively detect and quantify all peptides above an instruments limit of detection. Several software tools to analyze DIA data have been developed in recent years. However, several challenges still remain, like confidently identifying peptides, defining integration boundaries, dealing with interference for selected transitions, and scoring and filtering of peptide signals in order to control false discovery rates. In practice, a visual inspection of the signals is still required, which is impractical with large datasets. Avant-garde is a new tool to refine DIA (and PRM) by removing interfered transitions, adjusting integration boundaries and scoring peaks to control the FDR. Unlike other tools where MS runs are scored independently from each other, Avant-garde uses a novel data-driven scoring strategy. DIA signals are refined by learning from the data itself, using all measurements in all samples together to achieve the best optimization. We evaluated the performances of Avant-garde with a calibrated sample using spiked-in standards in a complex background, a phospho-enriched dataset (Abelin et al, 2016), and two complex hybrid proteome samples for benchmarking DIA software tools. The results clearly showed that Avant-garde is capable of improving the selectivity, accuracy, and reproducibility of the quantification results in very complex biological matrices. We have further shown that it can evaluate the suitability of a peak to be used for quantification reaching the same levels of selectivity, accuracy, and reproducibility obtained with manual validation.

bioinformatics↗

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↗

bioRxiv2018

Thesaurus: quantifying phosphoprotein positional isomers

Proteins can be phosphorylated at neighboring sites resulting in different functional states, and studying the regulation of these sites has been challenging. Here we present Thesaurus, a search engine that detects new positional isomers using site-specific fragment ions from parallel reaction monitoring and data independent acquisition mass spectrometry experiments. We apply Thesaurus to analyze phosphorylation events in the PI3K/AKT signaling pathway and show neighboring sites with distinct quantitative profiles, indicating regulation by different kinases.

bioinformatics↗

bioRxiv2018

Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

biochemistry↗

bioRxiv2018

Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation

Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteome, but unbiased mass-spectrometry measurements of chromatin-associated proteins remain difficult. In this work, we have combined facile nuclear sub-fractionation approaches with data-independent acquisition mass spectrometry to improve detection and quantification of nuclear proteins in human cells and tissues. Nuclei are isolated and subjected to a series of extraction conditions that enrich for nucleoplasm, euchromatin, heterochromatin and nuclear-membrane associated proteins. Using this approach, we can measure peptides from over 70% of the expressed nuclear proteome. As we are physically separating chromatin compartments prior to analysis, proteins can be assigned into functional chromatin environments to illuminate systems-wide nuclear protein dynamics. The integrity of nuclear sub-compartments were validated with immunofluorescence, which confirms the presence of key markers during chromatin extraction. We then apply this method to study the nuclear proteome-wide response to pharmacological degradation of the BET bromodomain proteins. BET degradation leads to widespread changes in chromatin composition, and we discover global HDAC1/2-mediated remodeling of chromatin previously bound by BET bromodomains. In summary, we have developed a technology for reproducible, comprehensive characterization of the nuclear proteome to observe the systems-wide nuclear protein dynamics.

systems biology↗

bioRxiv2018

Comprehensive peptide quantification for data independent acquisition mass spectrometry using chromatogram libraries

Data independent acquisition (DIA) mass spectrometry is a powerful technique that is improving the reproducibility and throughput of proteomics studies. We introduce a new experimental workflow that uses this technique to construct chromatogram libraries that capture fragment ion chromatographic peak shape and retention time for every detectable peptide in an experiment. These coordinates calibrate information in spectrum libraries or protein databases to a specific mass spectrometer and chromatography setup, and enable sensitive peptide detection in quantitative experiments. We also present EncyclopeDIA, a software tool for generating and searching chromatogram libraries, and demonstrate the performance of our workflow by quantifying proteins in human and yeast cells. We find that by exploiting calibrated retention time and fragmentation specificity in chromatogram libraries, EncyclopeDIA can detect and quantify >50% more peptides from DIA experiments than with DDA-based spectrum libraries alone.

biochemistry↗

bioRxiv2018

Novel phosphorylation states of the yeast spindle pole body

Phosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purified Saccharomyces cerevisiae SPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by a cdc4-1 temperature sensitive mutation, with particular interest in phosphorylation events on the {gamma}-tubulin small complex ({gamma}-TuSC). The cdc4-1 arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating {gamma}-TuSC. These sites were analyzed in vivo by fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally, in vivo analysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several {gamma}-TuSC phosphorylation sites influencing microtubule regulation.

biochemistry↗

bioRxiv2018

The human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Crosslinking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C-terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together our results indicate that the Ska complex can directly strengthen the kinetochore microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.\n\nSIGNIFICANCE STATEMENTMicrotubules are dynamic, tube-like structures that drive the segregation of duplicated chromosomes during cell division. The Ska complex is part of a molecular machine that forms force-bearing connections between chromosomes and microtubule ends. Depletion of the Ska complex destabilizes these connections and disrupts cell division. The Ska complex binds microtubules but it is unknown if it directly holds force at microtubules or indirectly stabilizes the connections. Here, we show that the Ska complex makes a direct force-bearing linkage with microtubule ends and assembles with another microtubule binding component, the Ndc80 complex, to strengthen its ability to withstand force. Our results suggest that the Ska and Ndc80 complexes work together to maintain the connections between chromosomes and microtubule ends.

biochemistry↗

bioRxiv2017

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations

Though the added value of proteomic measurements to gene expression profiling has been demonstrated, profiling of gene expression on its own remains the dominant means of understanding cellular responses to perturbation. Direct protein measurements are typically limited due to issues of cost and scale; however, the recent development of high-throughput, targeted sentinel mass spectrometry assays provides an opportunity for proteomics to contribute at a meaningful scale in high-value areas for drug development. To demonstrate the feasibility of a systematic and comprehensive library of perturbational proteomic signatures, we profiled 90 drugs (in triplicate) in six cell lines using two different proteomic assays -- one measuring global changes of epigenetic marks on histone proteins and another measuring a set of peptides reporting on the phosphoproteome -- for a total of more than 3,400 samples. This effort represents a first-of-its-kind resource for proteomics. The majority of tested drugs generated reproducible responses in both phosphosignaling and chromatin states, but we observed differences in the responses that were cell line-and assay-specific. We formalized the process of comparing response signatures within the data using a concept called connectivity, which enabled us to integrate data across cell types and assays. Furthermore, it facilitated incorporation of transcriptional signatures. Consistent connectivity among cell types revealed cellular responses that transcended cell-specific effects, while consistent connectivity among assays revealed unexpected associations between drugs that were confirmed by experimental follow-up. We further demonstrated how the resource could be leveraged against public domain external datasets to recognize therapeutic hypotheses that are consistent with ongoing clinical trials for the treatment of multiple myeloma and acute lymphocytic leukemia (ALL). These data are available for download via the Gene Expression Omnibus (accession GSE101406), and web apps for interacting with this resource are available at https://clue.io/proteomics.\n\nHighlightsO_LIFirst-of-its-kind public resource of proteomic responses to systematically administered perturbagens\nC_LIO_LIDirect proteomic profiling of phosphosignaling and chromatin states in cells for 90 drugs in six different cell lines\nC_LIO_LIExtends Connectivity Map concept to proteomic data for integration with transcriptional data\nC_LIO_LIEnables recognition of unexpected, cell type-specific activities and potential translational therapeutic opportunities\nC_LI

systems biology↗

bioRxiv2017

Time-resolved Interaction Proteomics of the Putative Scaffold Protein GIGANTEA in Arabidopsis thaliana

The large, plant-specific protein GIGANTEA (GI) is involved in many physiological processes, mediating rhythmic, post-translational regulation in part through circadian and light regulation of GI RNA expression. GI binds several proteins implicated in the circadian clock, the control of photoperiodic flowering, and abiotic stress responses, and has co-chaperone activity. By extension, further interaction partners might mediate the less well-understood roles of GI but the number and rhythmicity of these interactors is unknown. Here, we seek potential interactors in a time-specific manner, using quantitative proteomics from a time series study of transgenic Arabidopsis thaliana plants constitutively expressing an epitope-tagged GI protein. Previously-identified, direct and indirect interactors of GI were detected but no further F-box proteins related to known GI partners ZTL/FKF1/LKP2. The predominantly non-rhythmic, interacting proteins were implicated in protein folding or degradation, metabolism and chromatin modification, including a small set of partners shared with other clock-related proteins. A transcription factor homologue that we name CYCLING DOF FACTOR 6 (CDF6) was shown to interact both with GI and the ZTL/FKF1/LKP2 proteins and to control photoperiodic flowering. Our results indicate the biochemical pathways, beyond circadian and flowering regulation, that might be affected by GIGANTEAs rhythmic, post-translational control.\n\nSignificance StatementSignificance statement of up to two sentences of no more than 75 words total;\n\nThe GIGANTEA protein of Arabidopsis was known for circadian and flowering functions, mediated by the FKF1/LKP2/ZTL family of GI-interacting, F-box proteins, then for a co-chaperone activity of unknown scope. We performed time-resolved, interaction proteomics, identifying CDF6 (At1g26790) as a morning-specific GI interactor that controls flowering time. Unlike FKF1 and CDF proteins, most of the 240 candidate partners were not rhythmically enriched. They link GI to proteostasis and metabolic functions that might mediate GIs physiological functions.

plant biology↗

bioRxiv2017

Specter: linear deconvolution as a new paradigm for targeted analysis of data-independent acquisition mass spectrometry proteomics

Mass spectrometry with data-independent acquisition (DIA) has emerged as a promising method to greatly improve the comprehensiveness and reproducibility of targeted and discovery proteomics, in theory systematically measuring all peptide precursors within a biological sample. Despite the technical maturity of DIA, the analytical challenges involved in discriminating between peptides with similar sequences in convoluted spectra have limited its applicability in important cases, such as the detection of single-nucleotide polymorphisms and alternative site localizations in phosphoproteomics data. We have developed Specter, an open-source software tool that uses linear algebra to deconvolute DIA mixture spectra directly in terms of a spectral library, circumventing the problems associated with typical fragment correlation-based approaches. We validate the sensitivity of Specter and its performance relative to other methods by means of several complex datasets, and show that Specter is able to successfully analyze cases involving highly similar peptides that are typically challenging for DIA analysis methods.

bioinformatics↗