Biology subjects

Riffle, M.

Publications and source records attributed to Riffle, M..

24 records · Page 2Linked to original sources

bioRxiv2023

Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome

Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent [~]1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a "liquid biopsy," while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry of unfractionated plasma. We have developed an enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 L plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can collect results for >37,000 peptides from >4,000 plasma proteins with high precision. Using this analytical pipeline on a small cohort of patients with neurodegenerative disease and healthy age-matched controls, we discovered 204 proteins that differentiate (q-value < 0.05) patients with Alzheimers disease dementia (ADD) from those without ADD. Our method also discovered 310 proteins that were different between Parkinsons disease and those with either ADD or healthy cognitively normal individuals. Using machine learning we were able to distinguish between ADD and not ADD with a mean ROC AUC = 0.98 {+/-} 0.06.

biochemistry↗

bioRxiv2023

Evaluating the performance of the Astral mass analyzer for quantitative proteomics using data independent acquisition

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

biochemistry↗

bioRxiv2021

Discovery and visualization of uncharacterized drug-protein adducts using mass spectrometry.

Drugs are often metabolized to reactive intermediates that form protein adducts. Adducts can inhibit protein activity, elicit immune responses, and cause life threatening adverse drug reactions. The masses of reactive metabolites are frequently unknown, rendering traditional mass spectrometry-based proteomics incapable of adduct identification. Here, we present Magnum, an open-mass search algorithm optimized for adduct identification, and Limelight, a web-based data processing package for analysis and visualization of data from all existing algorithms. Limelight incorporates tools for sample comparisons and xenobiotic-adduct discovery. We validate our tools with two drug/protein combinations and apply our workflow to identify novel xenobiotic-protein adducts in CYP3A4. Our new methods and software enable accurate identification of xenobiotic-protein adducts with no prior knowledge of adduct masses or protein targets. Magnum outperforms existing tools in xenobiotic-protein adduct discovery, while Limelight fulfills a major need in the rapidly developing field of open-mass searching, which until now lacked comprehensive data visualization tools.

pharmacology and toxicology↗

bioRxiv2020

Survey of metaproteomics software tools for functional microbiome analysis.

To gain a thorough appreciation of microbiome dynamics, researchers characterize the functional role of expressed microbial genes/proteins. This can be accomplished through metaproteomics, which characterizes the protein complement of the microbiome. Several software tools exist for analyzing microbiomes at the functional level by measuring their combined proteome-level response to environmental perturbations. In this survey, we explore the performance of six available tools, so that researchers can make informed decisions regarding software choice based on their research goals. Tandem mass spectrometry-based proteomic data obtained from dental carie plaque samples grown with and without sucrose in paired biofilm reactors were used as representative data for this evaluation. Microbial peptides from one sample pair were identified by the X! Tandem search algorithm via SearchGUI and subjected to functional analysis using software tools including eggNOG-mapper, MEGAN6, MetaGOmics, MetaProteomeAnalyzer (MPA), ProPHAnE, and Unipept to generate functional annotation through Gene Ontology (GO) terms. Among these software tools, notable differences in functional annotation were detected after comparing differentially expressed protein functional groups. Based on the generated GO terms of these tools we performed a peptide-level comparison to evaluate the quality of their functional annotations. A BLAST analysis against the Universal Protein Knowledgebase revealed that the sensitivity and specificity of functional annotation differed between tools. For example, eggNOG-mapper mapped to the most number of GO terms, while Unipept generated the most precise GO terms. Based on our evaluation, metaproteomics researchers can choose the software according to their analytical needs and developers can use the resulting feedback to further optimize their algorithms. To make more of these tools accessible via scalable metaproteomics workflows, eggNOG-mapper and Unipept 4.0 were incorporated into the Galaxy platform.

bioinformatics↗

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↗