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Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

ABSTRACT: Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IMⁿ) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IMⁿ-MS experiments also show evidence of interconversion between compact and extended structures. IMⁿ-MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.

SUBMITTER: Eldrid C

PROVIDER: S-EPMC8172447 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

Eldrid Charles C Ben-Younis Aisha A Ujma Jakub J Britt Hannah H Cragnolini Tristan T Kalfas Symeon S Cooper-Shepherd Dale D Tomczyk Nick N Giles Kevin K Morris Mike M Akter Rehana R Raleigh Daniel D Thalassinos Konstantinos K

Journal of the American Society for Mass Spectrometry 20210518 6

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IM<sup>n</sup>) to be carried out. We d ...[more]

PMID: 34006100

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Project description:In recent years, ion mobility spectrometry-mass spectrometry (IMS-MS) has advanced the field of omics-based research, especially with the development of high-resolution platforms; however, these separations have generally been qualitative in nature. The rotationally averaged ion neutral collision cross section (CCS) is one of the only quantitative metrics available for aiding in characterizing biomolecules in IMS-MS. However, determining the CCS of an ion for multipass IMS systems, such as in cyclic ion mobility-mass spectrometry (cIMS-MS) and structures for lossless ion manipulations, has been challenging due to the lack of methods available for calculating CCS when more than a single pass is required for separation as well as the laborious nature of requiring calibrants and unknown compounds to be subjected to identical number of passes, which may not be possible in certain instances because of peak splitting, high levels of diffusion, etc. Herein, we present a general method that uses average ion velocities for calculating CCS values in cIMS-MS-based separations. Initially, we developed calibration curves using common CCS calibrants [i.e., tetra-alkylammonium salts, polyalanine, and hexakis(fluoroalkoxy)phosphazines] at different traveling wave (TW) conditions and the calculated cIMS CCS values were within ∼1% error or less compared to previously established drift tube IMS CCS measurements. Since it has been established that glycans can split into their α/β anomers, we utilized this method for two glycan species, 2α-mannobiose and melibiose. Both glycans were analyzed at the same TW conditions as the calibrants, and we observed anomer splitting at pathlengths of 20 m for 2α-mannobiose and 40 m for melibiose and thus assigned two unique CCS values for each glycan, which is the first time this has ever been done. We have demonstrated that the use of average ion velocities is a robust approach for obtaining CCS values with good agreement to CCS measurements from the previous literature and anticipate that this methodology can be applied to any IMS-MS platform that utilizes multipass separations. Our future work aims to incorporate this methodology for the development of a high-resolution CCS database to aid in the characterization of human milk oligosaccharides.

Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.

Dataset Information

Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

Publications

Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

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