Dataset Information

Differential solubilization and top-down proteomics with FAIMS control experiments applied to mouse brain

ABSTRACT: 5-35 mg brain tissue plugs from mouse left or right hemisphere were homogenized on ice with a pellet pestle homogenizer in a 1.5 mL LoBind Eppendorf tube after addition of 8 uL hexafluoroisopropanol (HFIP) / mg tissue. Once tissue was fully disrupted, an equivolume amount of homogenization buffer (HB, consisting of 8 M urea, 10 mM ammonium bicarbonate (ABC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 2 mM EDTA) was added and then vortexed for 1 min, before separation into soluble (referred to as "HFIP" in the datasets) and insoluble (referred to as "Urea" in the datasets) fractions. Samples were analyzed using a Waters NanoACQUITY UPLC system with mobile phases consisting of 0.2% FA in H2O (Mobile Phase A) and 0.2% FA in ACN (Mobile Phase B). Both trapping-precolumn (200 um i.d., 5-cm length) and analytical column (100 um i.d., 50-cm length) were slurry-packed with C2 packing material (5 um and 3 um for trap/analytical respectively, 300 A, Separation Methods Technology). Samples were loaded into a 10 uL loop, corresponding to 2.5 or 5 ug of loaded material and injected onto the trapping column with an isocratic flow of 1% B at 5 uL/min over 15 min for desalting. Separation was performed with a 1% to 50% B gradient over 160 min at 300 nL/min. For MS/MS analysis of proteins, the NanoACQUITY system was coupled to a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with the FAIMS Pro interface. Source parameters included electrospray voltage of 2.2 kV, transfer capillary temperature of 275 C, and ion funnel RF amplitude of 30%. FAIMS was set to standard resolution without supplementary user-controlled carrier gas flow and a dispersion voltage (DV) of -5 kV (equivalent to a dispersion field of -33.3 kV/cm), while the compensation voltage (CV) switched between three voltages (-50, -40, and -30) throughout data collection (referred to as "internal CV stepping"). The Fusion Lumos was set to "Intact Protein" application mode, and data was collected as full profile. Proteoform identification was performed with TopPIC version 1.5.4. Settings for TopPIC included a precursor window of 3 m/z, mass error tolerance of 15 ppm, a proteoform cluster error tolerance of 0.8 Da, a mass shift upper bound of 4000 Da and lower bound of -150 Da, and a maximum number of allowed unknown modifications of 1. All databases were scrambled to generate decoys which were concatenated during the search. A list of 12 dynamic modifications were provided during the open modification search to reduce the number of unknown mass shifts. Downstream data analysis was performed with TopPICR.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Vladislav Petyuk

PROVIDER: MSV000093727 | MassIVE | Mon Dec 25 20:28:00 GMT 2023

REPOSITORIES: MassIVE

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Publications

Discovery of Proteoforms Associated With Alzheimer's Disease Through Quantitative Top-Down Proteomics.

Fulcher James M JM Ives Ashley N AN Tasaki Shinya S Kelly Shane S SS Williams Sarah M SM Fillmore Thomas L TL Zhou Mowei M Moore Ronald J RJ Qian Wei-Jun WJ Paša-Tolić Ljiljana L Yu Lei L Oveisgharan Shahram S Bennett David A DA De Jager Philip L PL Petyuk Vladislav A VA

Molecular & cellular proteomics : MCP 20250505 6

The complex nature of Alzheimer's disease (AD) and its heterogenous clinical presentation has prompted numerous large-scale -omic analyses aimed at providing a global understanding of the pathophysiological processes involved. AD involves isoforms, proteolytic products, and posttranslationally modified proteins such as amyloid beta (Aβ) and microtubule-associated protein tau. Top-down proteomics directly measures these species and thus, offers a comprehensive view of pathologically relevant prot ...[more]

PMID: 40334744

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