MassIVE MSV000093728

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Large scale, quantitative top-down proteomic analysis of human frontal gyrus brain tissue samples in the context of Alzheimer's disease

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Description

15-35 mg brain tissue plugs derived from dorsal lateral prefrontal cortex (DLPFC) 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 "AB" in the datasets) and insoluble (referred to as "WO" 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. [doi:10.25345/C5DN4064X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: dementia ; brain ; FAIMS ; top-down proteomics ; intact protein ; TopPIC

Contact

Principal Investigators:
(in alphabetical order) 		Vladislav Petyuk, Pacific Northwest National Laboratory, United States
Submitting User: alchemistmatt

Publications

Fulcher JM, Ives AN, Tasaki S, Kelly SS, Williams SM, Fillmore TL, Zhou M, Moore RJ, Qian WJ, Paša-Toli? L, Yu L, Oveisgharan S, Bennett DA, De Jager PL, Petyuk VA.
Discovery of Proteoforms Associated with Alzheimer's Disease Through Quantitative Top-Down Proteomics.
Mol Cell Proteomics. Epub 2025 May 5.

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