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Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode

ABSTRACT: The native top-down mass spectrometry (nTDMS) platform is being applied in structural biology and discovery proteomics with great success, as it provides three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and subunit fragment masses that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While the intact mass data are readily converted deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment, essential for proteoform characterization, is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of subunit fragmentation data that result from nTDMS experiments on protein complexes and on diverse bioassemblies. We include an overview of strategies for navigating this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms.

INSTRUMENT(S): Q Exactive HF EMR

ORGANISM(S): Bos Taurus (ncbitaxon:9913) Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Neil Kelleher

PROVIDER: MSV000086404 | MassIVE | Tue Nov 03 11:00:00 GMT 2020

REPOSITORIES: MassIVE

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Project description:Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets. In MTBLS1333, 58 lipid mediator standards were used to study the lipid fragmentation, build optimal collision energy models, create in-silico spectral libraries and spike-in as internal standards. They were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Lipid fragment masses of mediators were validated with the Metlin database (https://metlin.scripps.edu/). Studies linked to this manuscript include;<a href='https://www.ebi.ac.uk/metabolights/mtbls1333' rel='noopener noreferrer' target='_blank'>MTBLS1333; LipidCreator: Collision Energy Optimization and Fragment Intensity Prediction for Lipid Mediators - Thermo QExactive HF</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1334' rel='noopener noreferrer' target='_blank'>MTBLS1334; LipidCreator: Collision Energy Optimization and Fragment Intensity Prediction for Lipid Mediators - Agilent QTof</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1369' rel='noopener noreferrer' target='_blank'>MTBLS1369; LipidCreator: Platelet isolation and stimulation - Targeted Phospho- and Glycerolipid Profiling</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1375' rel='noopener noreferrer' target='_blank'>MTBLS1375; LipidCreator: Targeted lipidomics analysis of Human Plasma samples and comparison with NIST SRM 1950 standard</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1376' rel='noopener noreferrer' target='_blank'>MTBLS1376; LipidCreator: Targeted lipidomics analysis of S. cerevisiae to determine true and false identification</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1381' rel='noopener noreferrer' target='_blank'>MTBLS1381; LipidCreator: Platelet isolation and stimulation - Targeted Lipid Mediator Profiling</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1382' rel='noopener noreferrer' target='_blank'>MTBLS1382; LipidCreator: Platelet isolation and stimulation - DIA Lipid Mediator Validation</a>

Project description:Here we present LipidCreator, a software that fully supports targeted lipidomics assay development. LipidCreator offers a comprehensive framework to compute MS/MS fragment masses for over 60 lipid classes. LipidCreator provides all functionalities needed to define fragments, manage stable isotope labeling, optimize collision energy and generate in silico spectral libraries. We validate LipidCreator assays computationally and analytically and prove that it is capable to generate large targeted experiments to analyze blood and to dissect lipid-signaling pathways such as in human platelets. In MTBLS1334, 55 lipid mediator standards were used to study the lipid fragmentation on the Agilent QTof 6545 MS platform (Agilent Technologies, Waldbronn, Germany), build optimal collision energy models and create an in-silico spectral library for use with LipidCreator. Lipid fragment masses of mediators were validated with the Metlin database (https://metlin.scripps.edu/). Studies linked to this manuscript include;<a href='https://www.ebi.ac.uk/metabolights/mtbls1333' rel='noopener noreferrer' target='_blank'>MTBLS1333; LipidCreator: Collision Energy Optimization and Fragment Intensity Prediction for Lipid Mediators - Thermo QExactive HF</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1334' rel='noopener noreferrer' target='_blank'>MTBLS1334; LipidCreator: Collision Energy Optimization and Fragment Intensity Prediction for Lipid Mediators - Agilent QTof</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1369' rel='noopener noreferrer' target='_blank'>MTBLS1369; LipidCreator: Platelet isolation and stimulation - Targeted Phospho- and Glycerolipid Profiling</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1375' rel='noopener noreferrer' target='_blank'>MTBLS1375; LipidCreator: Targeted lipidomics analysis of Human Plasma samples and comparison with NIST SRM 1950 standard</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1376' rel='noopener noreferrer' target='_blank'>MTBLS1376; LipidCreator: Targeted lipidomics analysis of S. cerevisiae to determine true and false identification</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1381' rel='noopener noreferrer' target='_blank'>MTBLS1381; LipidCreator: Platelet isolation and stimulation - Targeted Lipid Mediator Profiling</a><a href='https://www.ebi.ac.uk/metabolights/mtbls1382' rel='noopener noreferrer' target='_blank'>MTBLS1382; LipidCreator: Platelet isolation and stimulation - DIA Lipid Mediator Validation</a>

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Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode

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