Recommendations for reporting ion mobility Mass Spectrometry measurements.

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K ) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method.

Molecular Structures and Ion Mobility Cross Sections: Analysis of the Effects of He and N2 Buffer Gas.

An empirically observed correlation between ion mobility cross sections in helium and nitrogen buffer gases was examined as a function of temperature, molecular size, and shape. Experimental cross sections were determined for tetraglycine, bradykinin, angiotensin 2, melittin, and ubiquitin at 300 K and in the range from 80 to 550 K on home-built instruments and calculated by the projection superposition approximation (PSA) method. The PSA was also used to predict cross sections for larger systems such as human pancreatic alpha-amylase, concanavalin, Pichia pastoris lysyl oxidase, and Klebsiella pneumoniae acetolactate synthase.

Ion mobility analysis of molecular dynamics.

The combination of mass spectrometry and ion mobility spectrometry (IMS) employing a temperature-variable drift cell or a drift tube divided into sections to make IMS-IMS experiments possible allows information to be obtained about the molecular dynamics of polyatomic ions in the absence of a solvent. The experiments allow the investigation of structural changes of both activated and native ion populations on a timescale of 1-100 ms. Five different systems representing small and large, polar and nonpolar molecules, as well as noncovalent assemblies, are discussed in detail: a dinucleotide, a sodiated polyethylene glycol chain, the peptide bradykinin, the protein ubiquitin, and two types of peptide oligomers.

Factors contributing to the collision cross section of polyatomic ions in the kilodalton to gigadalton range: application to ion mobility measurements.

The projected superposition approximation (PSA) method was used to theoretically evaluate the factors contributing to the cross section measured in ion mobility experiments and to study how the significance of these factors varies with ion size from diglycine to a 1 μm oil droplet. Thousands of PSA calculations for ∼400 different molecules in the temperature range from 80 to 700 K revealed that the molecular framework made up of atomic hard spheres is, as expected, a major component of the cross section. However, the ion-buffer gas interaction is almost equally important for very small peptides, and although its significance decreases with increasing ion size, interaction is still a factor for megadalton ions.

Structural stability from solution to the gas phase: native solution structure of ubiquitin survives analysis in a solvent-free ion mobility-mass spectrometry environment.

The conformations of desolvated ubiquitin ions, lifted into the gas phase by electrospray ionization (ESI), were characterized by ion mobility spectrometry (IMS) and compared to the solution structures they originated from. The IMS instrument combining a two-meter helium drift tube with a quadrupole time-of-flight mass spectrometer was built in-house. Solutions stabilizing the native state of ubiquitin yielded essentially one family of tightly folded desolvated ubiquitin structures with a cross section matching the size of the native state (1000 Å(2)).

Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation.

Amyloid cascades that lead to peptide β-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with cellular machinery. The location and cause of the transformation from a natively unstructured assembly to the β-sheet oligomers found in all fibrils is important in understanding disease onset and the development of therapeutic agents.

Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease.

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates.

Conformational stability of Syrian hamster prion protein PrP(90-231).

Many transmissible spongiform encephalopathies (TSEs) are believed to be caused by a misfolded form of the normal cellular prion protein (PrP(C)) known as PrP(Sc). While PrP(Sc) is known to be exceptionally stable and resistant to protease degradation, PrP(C) has not shown these same unusual characteristics. However, using ion mobility spectrometry mass spectrometry (IMS-MS), we found evidence for at least one very stable conformation of a truncated form of recombinant PrP(C) consisting of residues 90-231, which resists unfolding in the absence of solvent at high injection energies and at temperatures in excess of 600 K.

Structural analysis of prion proteins by means of drift cell and traveling wave ion mobility mass spectrometry.

The prion protein (PrP) is implicitly involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). The conversion of normal cellular PrP (PrP(C)), a protein that is predominantly alpha-helical, to a beta-sheet-rich isoform (PrP(Sc)), which has a propensity to aggregate, is the key molecular event in prion diseases. During its short life span, PrP can experience two different pH environments; a mildly acidic environment, whilst cycling within the cell, and a neutral pH when it is glycosyl phosphatidylinositol (GPI)-anchored to the cell membrane.

The effect of calcium ions and peptide ligands on the relative stabilities of the calmodulin dumbbell and compact structures.

A combination of ion mobility and mass spectrometry methods was used to characterize the molecular shape of the protein calmodulin (CaM) and its complexes with calcium and a number of peptide ligands. CaM, a calcium-binding protein composed of 148 amino acid residues, was found by X-ray crystallography to occur both in a globular shape and in the shape of an extended dumbbell. Here, it was found, as solutions of CaM and CaM complexes were sprayed into the solvent-free environment of the mass spectrometer, that major structural features of the molecule and the stoichiometry of the units constituting a complex in solution were preserved in the desolvation process.

Systematic study of the structures of potassiated tertiary amino acids: salt bridge structures dominate.

The gas-phase structures of a series of potassiated tertiary amino acids have been systematically investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, ion mobility spectrometry (IMS), and computational modeling. The examined analytes comprise a set of five linear N,N-dimethyl amino acids derived from N,N-dimethyl glycine and three cyclic N-methyl amino acids including N-methyl proline. The number of methylene groups in either the alkyl chain of the linear members or in the ring of the cyclic members of the series is gradually varied.

Protonated arginine and protonated lysine: hydration and its effect on the stability of salt-bridge structures.

Using a mass spectrometer equipped with a drift cell, water binding energies of protonated arginine (ArgH+) and protonated lysine (LysH+) were determined in equilibrium experiments and supplementary calculations at the B3LYP/6-311++G** level of theory. The binding energy of the first water molecule was measured to be 10.3 and 10.

Amyloid beta-protein: experiment and theory on the 21-30 fragment.

The structure of the 21-30 fragment of the amyloid beta-protein (Abeta) was investigated by ion mobility mass spectrometry and replica exchange dynamics simulations. Mutations associated with familial Alzheimer's disease (E22G, E22Q, E22K, and D23N) of Abeta(21-30) were also studied, in order to understand any structural changes that might occur with these substitutions. The structure of the WT peptide shows a bend and a perpendicular turn in the backbone which is maintained by a network of D23 hydrogen bonding.

Hydration of protonated aromatic amino acids: phenylalanine, tryptophan, and tyrosine.

The first steps of hydration of the protonated aromatic amino acids phenylalanine, tryptophan, and tyrosine were studied experimentally employing a mass spectrometer equipped with a drift cell to examine the sequential addition of individual water molecules in equilibrium experiments and theoretically by a combination of molecular mechanics and electronic structure calculations (B3LYP/6-311++G**) on the three amino acid systems including up to five water molecules. It is found that both the ammonium and carboxyl groups offer good water binding sites with binding energies of the order of 13 kcal/mol for the first water molecule. Subsequent water molecules bind less strongly, in the range of 7-11 kcal/mol for the second through fifth water molecules.

Spermine binding to Parkinson's protein alpha-synuclein and its disease-related A30P and A53T mutants.

Aggregation of alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease (PD), is believed to progress through formation of a partially folded intermediate. Using nanoelectrospray ionization (nano-ESI) mass spectrometry combined with ion mobility measurements we found evidence for a highly compact partially folded family of structures for alpha-syn and its disease-related A53T mutant with net charges of -6, -7, and -8. For the other early onset PD mutant, A30P, this highly compact population was only evident when the protein had a net charge of -6.

Conformational evolution of ubiquitin ions in electrospray mass spectrometry: molecular dynamics simulations at gradually increasing temperatures.

Evidence from cross section data indicates that ubiquitin +13 ions lose their secondary and tertiary structure in mass spectrometric experiments. These transitions from the folded state into the near linear final structure occur at the experimental temperatures on time scales that are far too long for conventional molecular dynamics simulations. In this study, an approach to mass spectrometric unfolding processes is developed and a detailed application to an ubiquitin +13 ion system is presented.

Effects of familial Alzheimer's disease mutations on the folding nucleation of the amyloid beta-protein.

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid beta-protein (Abeta) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Abeta. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Abeta.

Interactions of the hormone oxytocin with divalent metal ions.

The interaction of the cyclic nonapeptide oxytocin (OT) with a number of alkaline earth and divalent transition metal ions (X(2+)) was examined employing mass spectrometry (MS) and ion mobility spectrometry (IMS) techniques in combination with molecular dynamics (MD) and density functional theory (DFT) calculations. Under acidic conditions it was found that OT exhibits an exceptionally strong affinity for all divalent metal ions resulting in strong [OT + X](2+) peaks in the mass spectrum. Under basic conditions only Cu(2+) and Ni(2+)-OT complexes were detected and these were singly, doubly, triply, or quadruply deprotonated.

Intermolecular interactions in biomolecular systems examined by mass spectrometry.

With the development of electrospray and matrix-assisted laser desorption ionization, mass spectrometry (MS) evolved into a powerful tool in the field of biochemistry. Whereas MS is primarily analytical in nature, an increasing number of MS research groups employ the method to address fundamental biochemical questions. Probing the interaction of noncovalently bound molecules in the mass spectrometer is one of the most interesting MS-based experiments possible today, with the potential of making a significant contribution to the basic understanding of the structure and function of biochemical complexes.

Hydration of mononucleotides.

The sequential addition of water molecules to protonated and deprotonated forms of the four mononucleotides dAMP, dCMP, dGMP, and dTMP was studied experimentally by equilibrium measurements using an electrospray mass spectrometer equipped with a drift cell and theoretically by computational methods including molecular modeling and density functional theory calculations. Experiments were carried out in positive and negative ion mode, and calculations included the protonated and deprotonated forms of the four nucleotides. For deprotonated anionic nucleotides the experimental enthalpies of hydration (DeltaH degrees n) were found to be similar for all four systems and varied between -10.

Elucidating amyloid beta-protein folding and assembly: A multidisciplinary approach.

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils.

Structure of the 21-30 fragment of amyloid beta-protein.

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent.

Amyloid beta-protein monomer structure: a computational and experimental study.

The structural properties of the Abeta42 peptide, a main constituent of the amyloid plaques formed in Alzheimer's disease, were investigated through a combination of ion-mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the -3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families.

Amyloid beta-protein: monomer structure and early aggregation states of Abeta42 and its Pro19 alloform.

The amyloid beta-protein (Abeta) is a seminal neuropathic agent in Alzheimer's disease (AD). Recent evidence points to soluble Abeta oligomers as the probable neurotoxic species. Among the naturally occurring Abeta peptides, the 42-residue form Abeta42 is linked particularly strongly with AD, even though it is produced at approximately 10% of the levels of the more abundant 40-residue form Abeta40.

Oxytocin-receptor binding: why divalent metals are essential.

Biologists have observed that the presence of divalent metal is essential for the binding of the hormone oxytocin (OT) to its cellular receptor. However, this interaction is not understood on the molecular level. Because conformation is a key factor controlling ligand binding in biomolecule systems, we have used ion mobility experiments and molecular modeling to probe the conformation of the oxytocin-zinc complex.