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.
Native ion mobility-mass spectrometry (IM-MS) has emerged as an information-rich technique for gas phase protein structure characterization; however, IM resolution is currently insufficient for the detection of subtle structural differences in large biomolecules. This challenge has spurred the development of collision-induced unfolding (CIU) which utilizes incremental gas phase activation to unfold a protein in order to expand the number of measurable descriptors available for native protein ions. Although CIU is now routinely used in native mass spectrometry studies, the interlaboratory reproducibility of CIU has not been established. Here we evaluate the reproducibility of the CIU data produced across three laboratories (University of Michigan, Texas A&M University, and Vanderbilt University). CIU data were collected for a variety of protein ions ranging from 8.6-66 kDa. Within the same laboratory, the CIU fingerprints were found to be repeatable with root mean square deviation (RMSD) values of less than 5%. Collision cross section (CCS) values of the CIU intermediates were consistent across the laboratories, with most features exhibiting an interlaboratory reproducibility of better than 1%. In contrast, the activation potentials required to induce protein CIU transitions varied between the three laboratories. To address these differences, three source assemblies were constructed with an updated ion activation hardware design utilizing higher mechanical tolerance specifications. The production-grade assemblies were found to produce highly consistent CIU data for intact antibodies, exhibiting high precision ion CCS and CIU transition values, thus opening the door to establishing databases of CIU fingerprints to support future biomolecular classification efforts.
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