Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification

Chemical cross-linking combined with mass spectrometry provides a method to study protein structures and interactions. The introduction of cleavable bonds in a cross-linker provides an avenue to decouple released peptide masses from their precursor species, greatly simplifying the downstream search, allowing for whole proteome investigations to be performed. Typically, these experiments have been challenging to carry out, often utilizing nonstandard methods to fully identify cross-linked peptides. Mango is an open source software tool that extracts precursor masses from chimeric spectra generated using cleavable cross-linkers, greatly simplifying the downstream search. As it is designed to work with chimeric spectra, Mango can be used on traditional high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers without the need for additional modifications. When paired with a traditional proteomics search engine, Mango can be used to identify several thousand cross-linked peptide pairs searching against the entire <i>Escherichia coli</i> proteome. Mango provides an avenue to perform whole proteome cross-linking experiments without specialized instrumentation or access to nonstandard methods

Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification

Chemical cross-linking combined with mass spectrometry provides a method to study protein structures and interactions. The introduction of cleavable bonds in a cross-linker provides an avenue to decouple released peptide masses from their precursor species, greatly simplifying the downstream search, allowing for whole proteome investigations to be performed. Typically, these experiments have been challenging to carry out, often utilizing nonstandard methods to fully identify cross-linked peptides. Mango is an open source software tool that extracts precursor masses from chimeric spectra generated using cleavable cross-linkers, greatly simplifying the downstream search. As it is designed to work with chimeric spectra, Mango can be used on traditional high-resolution tandem mass spectrometry (MS/MS) capable mass spectrometers without the need for additional modifications. When paired with a traditional proteomics search engine, Mango can be used to identify several thousand cross-linked peptide pairs searching against the entire <i>Escherichia coli</i> proteome. Mango provides an avenue to perform whole proteome cross-linking experiments without specialized instrumentation or access to nonstandard methods

<i>In Vivo</i> Protein Interaction Network Identified with a Novel Real-Time Cross-Linked Peptide Identification Strategy

Protein interaction topologies are critical determinants of biological function. Large-scale or proteome-wide measurements of protein interaction topologies in cells currently pose an unmet challenge that could dramatically improve understanding of complex biological systems. A primary impediment includes direct protein topology and interaction measurements from living systems since interactions that lack biological significance may be introduced during cell lysis. Furthermore, many biologically relevant protein interactions will likely not survive the lysis/sample preparation and may only be measured with <i>in vivo</i> methods. As a step toward meeting this challenge, a new mass spectrometry method called <b>Re</b>al-time <b>A</b>nalysis for <b>C</b>ross-linked peptide <b>T</b>echnology (ReACT) has been developed that enables assignment of cross-linked peptides “on-the-fly”. Using ReACT, 708 unique cross-linked (<5% FDR) peptide pairs were identified from cross-linked <i>E. coli</i> cells. These data allow assembly of the first protein interaction network that also contains topological features of every interaction, as it existed in cells during cross-linker application. Of the identified interprotein cross-linked peptide pairs, 40% are derived from known interactions and provide new topological data that can help visualize how these interactions exist in cells. Other identified cross-linked peptide pairs are from proteins known to be involved within the same complex, but yield newly discovered direct physical interactors. ReACT enables the first view of these interactions inside cells, and the results acquired with this method suggest cross-linking can play a major role in future efforts to map the interactome in cells

Large-Scale and Targeted Quantitative Cross-Linking MS Using Isotope-Labeled Protein Interaction Reporter (PIR) Cross-Linkers

Quantitative measurement of chemically cross-linked proteins is crucial to reveal dynamic information about protein structures and protein–protein interactions and how these are differentially regulated during stress, aging, drug treatment, and most perturbations. Previously, we demonstrated how quantitative in vivo cross-linking (CL) with stable isotope labeling of amino acids in cell culture (SILAC) enables both heritable and dynamic changes in cells to be visualized. In this work, we demonstrate the technical feasibility of proteome-scale quantitative in vivo CL–MS using isotope-labeled protein interaction reporter (PIR) cross-linkers and <i>E. coli</i> as a model system. This isotope-labeled cross-linkers approach, combined with Real-time Analysis of Cross-linked peptide Technology (ReACT) previously developed in our lab, enables the quantification of 941 nonredundant cross-linked peptide pairs from a total of 1213 fully identified peptide pairs in two biological replicate samples through comparison of MS¹ peak intensity of the light and heavy cross-linked peptide pairs. For targeted relative quantification of cross-linked peptide pairs, we further developed a PRM-based assay to accurately probe specific site interaction changes in a complex background. The methodology described in this work provides reliable tools for both large-scale and targeted quantitative CL–MS that is useful for any sample where SILAC labeling may not be practical

XLink-DB: Database and Software Tools for Storing and Visualizing Protein Interaction Topology Data

As large-scale cross-linking data becomes available, new software tools for data processing and visualization are required to replace manual data analysis. XLink-DB serves as a data storage site and visualization tool for cross-linking results. XLink-DB accepts data generated with any cross-linker and stores them in a relational database. Cross-linked sites are automatically mapped onto PDB structures if available, and results are compared to existing protein interaction databases. A protein interaction network is also automatically generated for the entire data set. The XLink-DB server, including examples, and a help page are available for noncommercial use at http://brucelab.gs.washington.edu/crosslinkdbv1/. The source code can be viewed and downloaded at https://sourceforge.net/projects/crosslinkdb/?source=directory

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

Cross-linking Measurements of the <i>Potato leafroll virus</i> Reveal Protein Interaction Topologies Required for Virion Stability, Aphid Transmission, and Virus–Plant Interactions

Protein interactions are critical determinants of insect transmission for viruses in the family <i>Luteoviridae</i>. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the <i>Potato leafroll virus</i> (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector–virus and plant–virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus

The methylome of <i>C</i>. <i>jejuni</i> F38011 contains 5 dominant methylation motifs.

<p>The methylation consensus sequences identified by PacBio with adenine methylations found in motifs 1, 2, 3, and 4 (motifs 1, 2 and 4 have a partner motif; RAATTY partner motif not shown) and cytosine methylation found in motif 5. Consensus sequences for each motif is represented as logos, where the height of each stack indicates conservation of sequence (bits) and the height of the symbols represent the relative frequency of the base. An asterisk below a base indicates the modified nucleotide in each consensus sequence. The consensus sequence on the circular genome is indicated with a black line. The numbers within each genome represent methylated sequences compared to the total number of each identified consensus sequence.</p