Next-generation proteomics for quantitative Jumbophage-bacteria interaction mapping

Fossati, Andrea; Mozumdar, Deepto; Kokontis, Claire; Mèndez-Moran, Melissa; Nieweglowska, Eliza; Pelin, Adrian; Li, Yuping; Guo, Baron; Krogan, Nevan J.; Agard, David A.; Bondy-Denomy, Joseph; Swaney, Danielle L.

doi:10.1038/s41467-023-40724-w

Cited by 16 publications

(13 citation statements)

References 65 publications

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“…We selected gp93 as a bona fide injected protein 20,21 to use as bait for AP-MS experiments hoping to reveal the early environment proximal to the phage genome. Fluorescent labeling experiments with other IB proteins revealed that gp90, gp95, and gp97 are also injected with the phage genome, while gp89 and gp162 remain in the head ( Supplementary Figure S1A ).…”

Section: Resultsmentioning

confidence: 99%

“…Prior work has demonstrated that the ΦKZ virion consists of up to ∼80 different virion proteins 19,20 . Of this list, three virion phage proteins gp92, gp97 and gp174 (Figure 1D, oval) appeared as gp93 interactors during infection (but not in gp93cyto) and are likely co-injected with gp93 and phage gDNA.…”

Section: Resultsmentioning

confidence: 99%

“…A previous mass spectrometry (MS) study with tailless virions reported six highly abundant virion proteins (gp89, 90, 93, 93, 95, 97, 162) that likely constitute the majority of this IB structure 19 . Our previous work using unbiased size exclusion chromatography-MS (SEC-MS) identified the IB interactome and immunoblotting demonstrated that 8 proteins are injected with the phage genome 20 . Additionally, fluorescence microscopy revealed that high copy gp93 is also deposited in the cell with the phage genome 21 .…”

Section: Introductionmentioning

confidence: 99%

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Defining the constituents and functions of a lipid-based jumbo phage compartment

Mozumdar,

Fossati,

Stevenson

et al. 2024

Preprint

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Viruses are vulnerable to cellular defenses at the start of the infection when a single copy genome has not yet initiated de novo viral protein synthesis. A family of jumbo phages related to phage ΦKZ, which infects Pseudomonas aeruginosa, uses a protein-based phage nucleus to protect the DNA during middle and late stages of infection, but how it is protected prior to phage nucleus assembly is unclear. To reveal the environment surrounding injected phage DNA, we determine which proteins interact with phage proteins injected with the phage genome. Here, we surprisingly identify host proteins related to membrane and lipid biology co-purifying with the injected phage protein. Staining of infected cells with lipophilic dyes revealed a clustering of host lipids co-localized with phage DNA and protein in an early phage infection (EPI) vesicle. Early gene expression is induced by the co-injected virion RNAP (vRNAP), with specific early proteins then associating with the EPI vesicle and ribosomes, likely to facilitate localized translation. As the infection progresses, the phage genome separates from the EPI vesicle and vRNAP, moving into the nascent proteinaceous phage nucleus. Enzymes involved in DNA replication and CRISPR/restriction immune nucleases notably do not localize to the EPI vesicle, supporting that DNA is not exposed at this early time point. We therefore propose that the EPI vesicle is rapidly constructed together with injected phage proteins, phage DNA, host lipids, and host membrane proteins to enable genome protection, early transcription, localized translation, and to ensure the faithful transfer of the single copy phage genome to the proteinaceous nucleus.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Defining the constituents and functions of a lipid-based jumbo phage compartment

Mozumdar,

Fossati,

Stevenson

et al. 2024

Preprint

Self Cite

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“…AP can also be added as an intermediate step to further reduce complexity or identify complexes with specific members . Andrea Fossati and colleagues recently applied CF-MS to identify jumbophage-bacteria PPIs . Jumbophages encode more than 300 proteins, and identifying PPIs using AP-MS is not necessarily practical.…”

Section: Ms-based Approaches In Ppi Identificationmentioning

confidence: 99%

“…Corresponding authors: refs , , , , , , , , and . First and corresponding authors: refs , , , , , , , and − .…”

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confidence: 99%

I’m Walking into Spiderwebs: Making Sense of Protein–Protein Interaction Data

Skawinski,

Shah

2024

J. Proteome Res.

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Protein−protein interactions (PPIs) are at the heart of the molecular landscape permeating life. Proteomics studies can explore this protein interaction landscape using mass spectrometry (MS). Thanks to their high sensitivity, mass spectrometers can easily identify thousands of proteins within a single sample, but that same sensitivity generates tangled spiderwebs of data that hide biologically relevant findings. So, what does a researcher do when she finds herself walking into spiderwebs? In a field focused on discovery, MS data require rigor in their analysis, experimental validation, or a combination of both. In this Review, we provide a brief primer on MS-based experimental methods to identify PPIs. We discuss approaches to analyze the resulting data and remove the proteomic background. We consider the advantages between comprehensive and targeted studies. We also discuss how scoring might be improved through AI-based protein structure information. Women have been essential to the development of proteomics, so we will specifically highlight work by women that has made this field thrive in recent years.

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Mapping protein–protein interactions by mass spectrometry

Liu,

Abad,

Chatterjee

et al. 2024

Mass Spectrometry Reviews

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Protein–protein interactions (PPIs) are essential for numerous biological activities, including signal transduction, transcription control, and metabolism. They play a pivotal role in the organization and function of the proteome, and their perturbation is associated with various diseases, such as cancer, neurodegeneration, and infectious diseases. Recent advances in mass spectrometry (MS)‐based protein interactomics have significantly expanded our understanding of the PPIs in cells, with techniques that continue to improve in terms of sensitivity, and specificity providing new opportunities for the study of PPIs in diverse biological systems. These techniques differ depending on the type of interaction being studied, with each approach having its set of advantages, disadvantages, and applicability. This review highlights recent advances in enrichment methodologies for interactomes before MS analysis and compares their unique features and specifications. It emphasizes prospects for further improvement and their potential applications in advancing our knowledge of PPIs in various biological contexts.

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Next-generation proteomics for quantitative Jumbophage-bacteria interaction mapping

Cited by 16 publications

References 65 publications

Defining the constituents and functions of a lipid-based jumbo phage compartment

Defining the constituents and functions of a lipid-based jumbo phage compartment

I’m Walking into Spiderwebs: Making Sense of Protein–Protein Interaction Data

Mapping protein–protein interactions by mass spectrometry

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