Increasing Collisional Activation of Protein Complexes Using Smaller Aperture Source Sampling Cones on a Synapt Q-IM-TOF Instrument with a Stepwave Source

Abstract

Quadrupole ion mobility time-of-flight (Q-IM-TOF) mass spectrometers have revolutionized investigation of native biomolecular complexes. High pressures in the sources of these instruments aid transmission of protein complexes through damping of kinetic energy by collisional cooling. As adducts are removed through collisional heating (declustering), excessive collisional cooling can prevent removal of nonspecific adducts from protein ions, leading to inaccurate mass measurements, broad mass spectral peaks, and obfuscation of ligand binding. We show that reducing the source pressure using smaller aperture source sampling cones (SC) in a Waters Synapt G2-Si instrument increases protein ion heating by decreasing collisional cooling, providing a simple way to enhance removal of adducted salts from soluble proteins (GroEL 14-mer) and detergents from a transmembrane protein complex (heptameric Staphylococcus aureus α-hemolysin, αHL). These experiments are supported by ion heating and cooling simulations which demonstrate reduced collisional cooling at lower source pressures. Using these easily swapped sample cones of different apertures is a facile approach to reproducibly extend the range of activation in Synapt-type instruments.

Approaches to Heterogeneity in Native Mass Spectrometry

“…For example, FT/GT methods can greatly facilitate identification of charge states for distinct, highly overlapped peaks in mass spectra, thus inputting the range of charge states thereby identified may greatly reduce artifacts of other deconvolution methods that perform best when the charge state range is confined to correct values. Experimental and instrumental improvements possible in the near future include development of robust inlet-based separation beyond liquid chromatography (such as capillary zone electrophoresis − ), next-generation nESI tip design (including reliable production of submicron emitters ,,,,,, and theta-glass emitters for rapid mixing of samples during the ESI process − ), and more efficient in-source desolvation. , …”

“…Experimental and instrumental improvements possible in the near future include development of robust inlet-based separation beyond liquid chromatography (such as capillary zone electrophoresis 139−141 ), next-generation nESI tip design (including reliable production of submicron emitters 5,6,118,306,555,556,558 and theta-glass emitters for rapid mixing of samples during the ESI process 599−601 ), and more efficient in-source desolvation. 210,602 In the more distant future, we also envision theoretical and instrument developments that reveal new types of information in native IM-MS data. For example, field alignment of biomolecular ions in IM-MS instruments may be used to separate ions based on structural differences not easily observed in experiments on current low-field IM-MS instruments.…”

Native Mass Spectrometry of Membrane Proteins

“…Nonionic detergents are often preferable for solubilization of membrane proteins because they are usually less denaturing than ionic detergents. , Nonionic detergents are also desirable for MS because they minimize ion suppression and are generally easier to remove in the gas phase. Prell and co-workers have recently developed a novel Gabor transform data analysis approach that enables measurement of the membrane protein oligomeric state in highly adducted Fos-14, a zwitterionic detergent. , However, these Fos-choline micelles can be denaturing for some membrane proteins and should be used carefully.…”

“…Prell and co-workers have recently developed a novel Gabor transform 40 data analysis approach that enables measurement of the membrane protein oligomeric state in highly adducted Fos-14, a zwitterionic detergent. 41,42 However, these Fos-choline micelles can be denaturing for some membrane proteins and should be used carefully.…”

Modified Ion Source for the Improved Collisional Activation of Protein Complexes

“…Collisional activation is often necessary to remove retained adducts, though in some highly purified samples, high resolution is achieved without collisional activation. For membrane protein complexes, collisional activation is necessary to even observe the protein complex out of the detergent micelle. , Optimization of the pressures in the source region of quadrupole time-of-flight (Q-TOF) instruments can be used to increase the amount of activation in the source region necessary for analysis of protein complexes. ,− To achieve high resolution for protein complexes in Orbitrap systems, ions are trapped in the higher energy collisional dissociation (HCD) cell as opposed to the C-trap, where the increased pressure improves desolvation. , In addition to trapping in the HCD cell, ions can also be trapped in the source region so that the desolvation occurs prior to the quadrupole mass filter. , …”