David E. Clemmer

Ion Mobility Measurements and their Applications to Clusters and Biomolecules

Ion mobility measurements can be used to obtain structural information for large polyatomic ions in the gas phase. The methods are flexible and can be applied to a wide range of chemical systems. This article reviews the development of these methods and discusses recent applications to complex ions such as atomic clusters and large biomolecules.

Biomolecule Analysis by Ion Mobility Spectrometry

Although nonnative protein conformations, including intermediates along the folding pathway and kinetically trapped misfolded species that disfavor the native state, are rarely isolated in the solution phase, they are often stable in the gas phase, where macromolecular ions from electrospray ionization can exist in varying charge states. Differences in the structures of nonnative conformations in the gas phase are often large enough to allow different shapes and charge states to be separated because of differences in their mobilities through a gas. Moreover, gentle collisional activation can be used to induce structural transformations. These new structures often have different mobilities. Thus, there is the possibility of developing a multidimensional separation that takes advantage of structural differences of multiple stable states. This review discusses how nonnative states differ in the gas phase compared with solution and presents an overview of early attempts to utilize and manipulate structures in order to develop ion mobility spectrometry as a rapid and sensitive technique for separating complex mixtures of biomolecules prior to mass spectrometry.

HIGH-RESOLUTION ION MOBILITY MEASUREMENTS

Gas phase ion mobility measurements can resolve structural isomers for polyatomic ions and provide information about their geometries. A new experimental apparatus for performing high-resolution ion mobility measurements is described. The apparatus consists of a pulsed laser vaporization/desorption source coupled through an ion gate to a 63-cm-long drift tube. The ion gate is a critical component that prevents the diffusion of neutral species from the source into the drift tube. Ions travel along the drift tube under the influence of a uniform electric field. At the end of the drift tube some of the ions exit through a small aperture. They are focused into a quadrupole mass spectrometer, where they are mass analyzed, and then detected by an off-axis collision dynode and by dual microchannel plates. The apparatus is operated with a drift voltage of up to 14 000 V and a helium buffer gas pressure of around 500 Torr. The resolving power for ion mobility measurements is over an order of magnitude higher than ...

Activation of hydrogen and methane by thermalized FeO+ in the gas phase as studied by multiple mass spectrometric techniques

Abstract The ion-molecule reactions of thermalized iron-oxide cation FeO + with dihydrogen and methane have been studied by three different experimental techniques: Fourier transform ion cyclotron resonance (ICR), guided ion beam (GIB), and selected-ion flow tube (SIFT) mass spectrometry. Although these studies agree in a qualitative sense, i.e., FeO + brings about activation of H 2 and CH 4 with quite low efficiencies, there exists a considerable quantitative divergence as far as rate constants and branching ratios are concerned. The sources of error in these three related, but yet different experimental techniques are analyzed and critically reviewed. This error analysis brings the data to internal consistency with each other, once an accurate reference is used for calibration. In general, the rate constants obtained with the SIFT apparatus appear as the most accurate ones, while those obtained under ICR conditions are slightly too large, and the rate constants determined with the GIB instrument are somewhat lower than SIFT. However, the branching ratios for the formation of Fe + and FeOH + in the reaction of FeO + with methane are subject to more subtle effects. In the SIFT apparatus, termolecular stabilization of the intermediates causes differences from the ICR and GIB measurements, which were obtained under single-collision conditions.

Conformer-Dependent Proton-Transfer Reactions of Ubiquitin Ions

The conformations of ubiquitin ions before and after being exposed to proton transfer reagents have been studied by using ion mobility/mass spectrometry techniques. Ions were produced by electrospray ionization and exposed to acetone, acetophenone, n-butylamine, and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene. Under the conditions employed, the +4 to +13 charge states were formed and a variety of conformations, which we have characterized as compact, partially folded, and elongated, have been observed. The low charge state ions have cross sections that are similar to those calculated for the crystal conformation. High charge states favor unfolded conformations. The ion mobility distributions recorded after ions have been exposed to each base show that the lowest charge state that is formed during proton-transfer reactions favors a compact conformation. More open conformations are observed for the higher charge states that remain after reaction. The results show that for a given charge state, the apparent gas-phase acidities of the different conformations are ordered as compact < partially folded < elongated.

A computational approach toward label-free protein quantification using predicted peptide detectability

We propose here a new concept of peptide detectability which could be an important factor in explaining the relationship between a proteins quantity and the peptides identified from it in a high-throughput proteomics experiment. We define peptide detectability as the probability of observing a peptide in a standard sample analyzed by a standard proteomics routine and argue that it is an intrinsic property of the peptide sequence and neighboring regions in the parent protein. To test this hypothesis we first used publicly available data and data from our own synthetic samples in which quantities of model proteins were controlled. We then applied machine learning approaches to demonstrate that peptide detectability can be predicted from its sequence and the neighboring regions in the parent protein with satisfactory accuracy. The utility of this approach for protein quantification is demonstrated by peptides with higher detectability generally being identified at lower concentrations over those with lower detectability in the synthetic protein mixtures. These results establish a direct link between protein concentration and peptide detectability. We show that for each protein there exists a level of peptide detectability above which peptides are detected and below which peptides are not detected in an experiment. We call this level the minimum acceptable detectability for identified peptides (MDIP) which can be calibrated to predict protein concentration. Triplicate analysis of a biological sample showed that these MDIP values are consistent among the three data sets.

A database of 660 peptide ion cross sections: Use of intrinsic size parameters for bona fide predictions of cross sections

An ion trap/ion mobility/time-of-flight mass spectrometry technique has been used to measure collision cross sections for 660 peptide ions generated by tryptic digestion of 34 common proteins. Measured cross sections have been compiled into a database that contains peptide molecular weight and sequence information. The database is used to generate average intrinsic contributions to cross section (size parameters) for different amino acid residues by solving systems of equations that relate the unknown contributions of individual residues to the sequences and cross sections of database peptides. Size parameters are combined with information about amino acid composition to calculate cross sections for database peptides. Bona fide cross section predictions (made prior to measurement) for peptides observed in tryptic digests of sperm whale myoglobin and yeast enolase are made. Eight of 10 predicted cross sections are within 2% of the experimental values and all 10 are within 3.2%. The utility of size parameters for cross section prediction is explored and discussed.

Profiling of Human Serum Glycans Associated with Liver Cancer and Cirrhosis by IMS-MS

Aberrant glycosylation of human glycoproteins is related to various physiological states, including the onset of diseases such as cancer. Consequently, the search for glycans that could be markers of diseases or targets of therapeutic drugs has been intensive. Here, we describe a high-throughput ion mobility spectrometry/mass spectrometry analysis of N-linked glycans from human serum. Distributions of glycans are assigned according to their m/z values, while ion mobility distributions provide information about glycan conformational and isomeric composition. Statistical analysis of data from 22 apparently healthy control patients and 39 individuals with known diseases (20 with cirrhosis of the liver and 19 with liver cancer) shows that ion mobility distributions for individual m/z ions appear to be sufficient to distinguish patients with liver cancer or cirrhosis. Measurements of glycan conformational and isomeric distributions by IMS-MS may provide insight that is valuable for detecting and characterizing disease states.

Ion Mobility Spectrometry/Mass Spectrometry Snapshots for Assessing the Molecular Compositions of Complex Polymeric Systems

The synthesis of increasingly complex polymers has created daunting, sometimes insurmountable problems for their chemical analysis. The importance is magnified by outsourcing of production and their use in consumer products, including medical devices and food storage, and therefore requires a new generation of technology for quality assurance. Here, we report capturing subtle differences at the molecular level in complex polymer mixtures nearly instantaneously using a prototype multidimensional ion mobility spectrometry/mass spectrometry spectrometry instrument. Bulk activation/fragmentation strategies reported here provide signatures of structural characteristics that permit effortless recognition of minor differences in blends and copolymers, even as structural isomers and from a quantitative perspective. The data displayed as a pictorial snapshot provide a visual pattern that is sufficiently distinctive that computer-aided pattern recognition can be used to address process control and regulatory issues.

Number of solution states of bradykinin from ion mobility and mass spectrometry measurements.

Ion mobility and mass spectrometry measurements have been used to examine the populations of different solution structures of the nonapeptide bradykinin. Over the range of solution compositions studied, from 0:100 to 100:0 methanol:water and 0:100 to 90:10 dioxane:water, evidence for 10 independent populations of bradykinin structures in solution is found. In some solutions as many as eight structures may coexist. The solution populations are substantially different than the gas-phase equilibrium distribution of ions, which exhibits only three distinct states. Such a large number of coexisting structures explains the inability of traditional methods of characterization such as nuclear magnetic resonance spectroscopy and crystallography to determine detailed structural features for some regions of this peptide.