First-Principles Collision Cross Section Measurements of Large Proteins and Protein Complexes

Abstract

Rotationally averaged collision cross section (CCS) values for a series of proteins and protein complexes ranging in size from 8.6 to 810 kDa are reported. The CCSs were obtained using a native electrospray ionization drift tube ion mobility-Orbitrap mass spectrometer specifically designed to enhance sensitivity while having high-resolution ion mobility and mass capabilities. Periodic focusing (PF)-drift tube (DT)-ion mobility (IM) provides first-principles determination of the CCS of large biomolecules that can then be used as CCS calibrants. The experimental, first-principles CCS values are compared to previously reported experimentally determined and computationally calculated CCS using projected superposition approximation (PSA), the Ion Mobility Projection Approximation Calculation Tool (IMPACT), and Collidoscope. Experimental CCS values are generally in agreement with previously reported CCSs, with values falling within ∼5.5%. In addition, an ion mobility resolution (CCS centroid divided by CCS fwhm) of ∼60 is obtained for pyruvate kinase (MW ∼ 233 kDa); however, ion mobility resolution for bovine serum albumin (MW ∼ 68 kDa) is less than ∼20, which arises from sample impurities and underscores the importance of sample quality. The high resolution afforded by the ion mobility-Orbitrap mass analyzer provides new opportunities to understand the intricate details of protein complexes such as the impact of post-translational modifications (PTMs), stoichiometry, and conformational changes induced by ligand binding.

Exploring the conformational landscape and stability of Aurora A using ion-mobility mass spectrometry and molecular modelling

“…3). This difference of 5-10% compared to the experimentally determined values is similar to the differences previously observed between experimental and IMPACT computed CCS values for other proteins (72). The distributions of exemplar DFG-in (1OL7), DFG-inter (5L8K) and DFG-out (6HJK) structures are virtually indistinguishable, whilst the higher CCS values are almost exclusively from the 4C3P (DFG-in, A-loop open) simulations (Supp.…”

Variable-Temperature Electrospray Ionization for Temperature-Dependent Folding/Refolding Reactions of Proteins and Ligand Binding

“…The TEC (Peltier) vT-ESI source was designed for easy implementation on different MS platforms. These include a number of mass spectrometersthe ThermoFisher Orbitraps, including those equipped with REIS and FT-IMS capabilities, ,,− the Agilent 6560 IM-QTOF and 6545 XT, and the Waters SYNAPT instruments equipped with TWIMS. The performance metrics of the three-stage TEC vT-ESI device make it possible to rapidly acquire reproducible melting curves for static spray capillaries over the temperature range of 5–98 °C.…”

“…Additional information on the data processing can be found in the Supporting Information. The IM-MS data were collected using a 1.5 m drift-tube Fourier-transform ion mobility-UHMR Orbitrap, as described previously. ,− A-state ubiquitin IM-MS data were collected on a Waters SYNAPT G2 modified with the newly designed vT-ESI source. CCS calibration was performed using the method described by Ruotolo et al…”

Supercharging Prions via Amyloid‐Selective Lysine Acetylation

“…Serum albumin was the most acetylated native protein that we studied and is aspecial case.Wecould not integrate all ions to determine accurate weighted averages of numbers of acetylated lysines because the mass spectra of HSA are reportedly difficult to deconvolute due to persistent impurities. [32] We could, however, calculate the number of acetylated lysine on the most abundant ions.O ft he 60 lysine in HSA, we estimate that BT acetylated between 3-34 Lys (median % 15 Lys), SB acetylated 1-43 Lys(median % 15 Lys), and Py acetylated 12-45 Lys(median % 25 Lys). Unacetylated HSA was not detected.…”