Browsing by Author "Moritz, Robert L."

Now showing 1 - 5 of 5
  • Thumbnail Image
    Item
    Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry
    (American Chemical Society, 2024-06-04) Jiang, Yuming; Rex, Devasahayam Arokia Balaya; Schuster, Dina; Neely, Benjamin A.; Rosano, Germán L.; Volkmar, Norbert; Momenzadeh, Amanda; Peters-Clarke, Trenton M.; Egbert, Susan B.; Kreimer, Simion; Doud, Emma H.; Crook, Oliver M.; Yadav, Amit Kumar; Vanuopadath, Muralidharan; Hegeman, Adrian D.; Mayta, Martín L.; Duboff, Anna G.; Riley, Nicholas M.; Moritz, Robert L.; Meyer, Jesse G.; Biochemistry and Molecular Biology, School of Medicine
    Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this Review will serve as a handbook for researchers who are new to the field of bottom-up proteomics.
  • Thumbnail Image
    Item
    Comprehensive Overview of Bottom-Up Proteomics using Mass Spectrometry
    (ArXiv, 2023-11-13) Jiang, Yuming; Rex, Devasahayam Arokia Balaya; Schuster, Dina; Neely, Benjamin A.; Rosano, Germán L.; Volkmar, Norbert; Momenzadeh, Amanda; Peters-Clarke, Trenton M.; Egbert, Susan B.; Kreimer, Simion; Doud, Emma H.; Crook, Oliver M.; Yadav, Amit Kumar; Vanuopadath, Muralidharan; Mayta, Martín L.; Duboff, Anna G.; Riley, Nicholas M.; Moritz, Robert L.; Meyer, Jesse G.; Biochemistry and Molecular Biology, School of Medicine
    Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics.
  • Thumbnail Image
    Item
    Global Release of Translational Repression Across Plasmodium’s Host-to-Vector Transmission Event
    (bioRxiv, 2024-03-16) Rios, Kelly T.; McGee, James P.; Sebastian, Aswathy; Moritz, Robert L.; Feric, Marina; Absalon, Sabrina; Swearingen, Kristian E.; Lindner, Scott E.; Pharmacology and Toxicology, School of Medicine
    Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.
  • Thumbnail Image
    Item
    Recent Progress in Lyme Disease and Remaining Challenges
    (Frontiers Media, 2021-08-18) Bobe, Jason R.; Jutras, Brandon L.; Horn, Elizabeth J.; Embers, Monica E.; Bailey, Allison; Moritz, Robert L.; Zhang, Ying; Soloski, Mark J.; Ostfeld, Richard S.; Marconi, Richard T.; Aucott, John; Ma’ayan, Avi; Keesing, Felicia; Lewis, Kim; Mamoun, Choukri Ben; Rebman, Alison W.; McClune, Mecaila E.; Breitschwerdt, Edward B.; Reddy, Panga Jaipal; Maggi, Ricardo; Yang, Frank; Nemser, Bennett; Ozcan, Aydogan; Garner, Omai; Di Carlo, Dino; Ballard, Zachary; Joung, Hyou-Arm; Garcia-Romeu, Albert; Griffiths, Roland R.; Baumgarth, Nicole; Fallon, Brian A.; Microbiology and Immunology, School of Medicine
    Lyme disease (also known as Lyme borreliosis) is the most common vector-borne disease in the United States with an estimated 476,000 cases per year. While historically, the long-term impact of Lyme disease on patients has been controversial, mounting evidence supports the idea that a substantial number of patients experience persistent symptoms following treatment. The research community has largely lacked the necessary funding to properly advance the scientific and clinical understanding of the disease, or to develop and evaluate innovative approaches for prevention, diagnosis, and treatment. Given the many outstanding questions raised into the diagnosis, clinical presentation and treatment of Lyme disease, and the underlying molecular mechanisms that trigger persistent disease, there is an urgent need for more support. This review article summarizes progress over the past 5 years in our understanding of Lyme and tick-borne diseases in the United States and highlights remaining challenges.
  • Thumbnail Image
    Item
    Widespread release of translational repression across Plasmodium’s host-to-vector transmission event
    (Public Library of Science, 2025-01-08) Rios, Kelly T.; McGee, James P.; Sebastian, Aswathy; Gedara, Sanjaya Aththawala; Moritz, Robert L.; Feric, Marina; Absalon, Sabrina; Swearingen, Kristian E.; Lindner, Scott E.; Pharmacology and Toxicology, School of Medicine
    Malaria parasites must respond quickly to environmental changes, including during their transmission between mammalian and mosquito hosts. Therefore, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. While the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, changes in the spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that ~200 transcripts are released for translation soon after fertilization, including those encoding essential functions. Moreover, we identified that many transcripts remain repressed beyond this point. TurboID-based proximity proteomics of the DOZI/CITH/ALBA regulatory complex revealed substantial spatial and/or compositional changes across this transmission event, which are consistent with recent, paradigm-shifting models of translational control. Together, these data provide a model for the essential translational control mechanisms that promote Plasmodium's efficient transmission from mammalian host to mosquito vector.