Resolution and structural transitions of elongated states of ubiquitin

Deprotonation reactions of multiply charged protein ions have been studied by introducing volatile reference bases at atmospheric pressure between an electrosonic spray ionization (ESSI) source and the inlet of a mass spectrometer. Apparent gas-phase basicities (GB app ) of different charge states of protein ions were determined by a bracketing approach. The results pp obtained depend on the conformation of the protein ions in the gas phase, which is linked to the type of buffer used (denaturing or nondenaturing [1] rapidly became established as the method of choice for the production of large biomolecular ions in the gas phase. For proteins, ESI leads to the formation of multiply charged ions, both in the positive and negative ion modes. The charge-state distribution is directly related to the conformation of the macromolecular ions [2] and the proton transfer reactions in the gas phase [3]. Thermodynamic information, such as values for the gas-phase acidity/basicity (GA/GB), is essential to the understanding of the proton transfer processes and generation of ions by ESI in general. The intrinsic gas-phase basicity of a [M ϩ (n Ϫ 1)H] (n Ϫ 1)ϩ ion or the gas-phase acidity of a [M ϩ nH] nϩ ion can be defined as the free enthalpy change ⌬G 0 of the reaction:Since this reaction proceeds with a reverse activation barrier, due to Coulombic repulsion between the two charges product ions, the terms "apparent gas-phase acidity" (GA app ) and "apparent gas-phase basicity" (GB app ) are more appropriate. The GA app is defined as the sum of the ⌬G and the energy of the activation barrier, considered as a negative term [4].Determination of GB app s of peptides and proteins provides important information which is analogous to measurements of pKa in solution. For example, pKa values are greatly affected by intramolecular interactions, and the conversion of the denatured to the native form maximizes these interactions. Similarly, GB app values will also reflect the conformation of the ions in the gas phase.For more than 10 years, research groups have tried to determine apparent gas-phase basicities of peptides and proteins. For small peptides, like bradykinin and its des-arginine analogues, the kinetic method can be directly used to determine GB app values [5,6]. The energy of the activation barrier was obtained by measuring the kinetic energy release to calculate the intrinsic GB of two different charge states. Unfortunately, this method is quite difficult to transfer to multiply charged protein ions.A bracketing technique, consisting of evaluating the qualitative or quantitative rate of proton transfer between a neutral volatile base and protein ions was introduced to measure GB app values of multiply charges ions [4,7,8]. In terms of instrumentation, different systems can be used to observe the deprotonation reactions. High-pressure mass spectrometry (HPMS) experiments were designed by the group of Kebarle to study cluster formation from small organic or inorganic ions, and water in particular [9,10]. This apparatus was...

Section: Human Carbonic Anhydrase II and Creatine Kinasementioning

confidence: 99%

Investigation of deprotonation reactions on globular and denatured proteins at atmospheric pressure by ESSI-MS

Touboul

Jecklin

Zenobi

2008

“…C urrent research using electrospray ionization (ESI) coupled to mass spectrometry (MS) and ion mobility spectrometry (IMS) has demonstrated the ability of these inherently gas-phase techniques to provide details about the structures of large macromolecules [1][2][3][4]. Protein function determination is critical for the identification of potential drug targets, an understanding of diseases, and for the elucidation of biochemical pathways; structural studies aid in this assessment of protein function [1,3,5].…”

mentioning

confidence: 99%

Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility

Kaddis

Lomeli

Yin

et al. 2007

146

149

Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm 3 . Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. (J Am Soc Mass Spectrom 2007, 18, 1206 -1216

“…Initial experiments and instrument characteristics are described, including use of a new ion trap controller and software to control the entire instrument. Ubiquitin is used as a test compound to compare the new results with the extensive previous studies of this protein [21,[47][48][49][50][51][52].…”

mentioning

confidence: 99%

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

Zhao

Soyk

Schieffer

et al. 2009

This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin ϩ7 ions are converted to lower charge states down to ϩ1; the ions in lower charge states tend to be in compact conformations with cross sections down to ϳ880 Å 2 . The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin ϩ7 and ϩ6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described. Ion mobility (IM) [4,5] has become a very useful technique for analysis of biological ions in the gas phase [6]. IM provides information about ion size and structure [7], as it rapidly separates ions based on collision cross-section, rather than just m/z ratio. The use of IM to disperse a mixture of ions in time before analysis via a time-of-flight (TOF) MS, i.e., nested drift (flight) time measurements, is an important recent advance. These experiments were pioneered by Clemmer and coworkers in the mid-1990s [8], and have now been used by several other groups [9 -13]. The recent Synapt ESI-IMS-MS by Waters Corp. provides a commercially available instrument for gas-phase ion conformation studies by IM using a novel traveling wave approach [9].In a series of instrumental designs, the Clemmer group has made various modifications to the initial ESI-IM-TOF, including the insertion of a collision cell between the IM drift tube and the TOF for mobility labeling experiments [14,15], and the addition of an IT before the mobility drift tube to improve the duty cycle from the continuous ESI source [16,17]. One publication demonstrated MS/MS capabilities with an ion trap before IM-TOF [18], but the entire instrument was not under computer control. Therefore, only relatively simple experiments were possible.IM has been used to analyze the products of ionmolecule reactions [19], including proton transfer [20,21], H/D exchange [22], and solvation [23][24][25][26]. In these studies, the desired reactions take place either in the atmospheric pressure ion source interface region or in the drift tube itself. Thus, only short reaction times and certain reagent ions can be used. In addition, performing reactions in the IM cell can make spe...