Ion mobility spectrometry provides information about molecular structures of ions. Hence, high resolving power allows separation of isomers which is of major interest in several applications. In this work, we couple our high-resolution ion mobility spectrometer (IMS) with a resolving power of Rp?=?100 to a time-of-flight mass spectrometer (TOF-MS). Besides, the benefit of an increased resolving power such an IMS-MS also helps analyzing and understanding the ionization processes in IMS. Usually, the coupling between IMS and TOF-MS is realized by synchronizing data acquisition of the IMS and MS resulting in two-dimensional data containing ion mobility and mass spectra. However, due to peak widths of less than 100 μs in our high-resolution IMS, this technique is not practicable due to significant peak broadening during the ion transfer into the MS and an insufficient data acquisition rate of the MS. Thus, a novel but simple interface between the IMS and MS has been designed which minimizes ion losses, allows recording of ion mobility at full IMS resolving power, and enables a shuttered transmission of ions into the MS. The interface is realized by replacing the Faraday plate used in IMS by a Faraday grid that is shielded by two additional aperture grids. For demonstration, positive product ions of benzene, toluene, and m-xylene in air are investigated. The IMS is equipped with a radioactive 3H source. Besides the well-known product ions M+ and M·NO+, a dimer ion is also observed for benzene and toluene, consisting of two molecules and three further hydrogen atoms.
Corona discharge ionization sources are often used in ion mobility spectrometers (IMS) when a non-radioactive ion source with high ion currents is required. Typically, the corona discharge is followed by a reaction region where analyte ions are formed from the reactant ions. In this work, we present a simple yet sufficiently accurate model for predicting the ion current available at the end of this reaction region when operating at reduced pressure as in High Kinetic Energy Ion Mobility Spectrometers (HiKE-IMS) or most IMS-MS instruments. It yields excellent qualitative agreement with measurement results and is even able to calculate the ion current within an error of 15%. Additional interesting findings of this model are the ion current at the end of the reaction region being independent from the ion current generated by the corona discharge and the ion current in High Kinetic Energy Ion Mobility Spectrometers (HiKE-IMS) growing quadratically when scaling down the length of the reaction region.
The rising profile of ion mobility spectrometry (IMS) in proteomics has driven the efforts to predict peptide cross-sections. In the simplest approach, these are derived by adding the contributions of all amino acid residues and post-translational modifications (PTMs) defined by their intrinsic size parameters (ISPs). We show that the ISPs for PTMs can be calculated from properties of constituent atoms, and introduce the “impact scores” that govern the shift of cross-sections from the central mass-dependent trend for unmodified peptides. The ISPs and scores tabulated for 100 more common PTMs enable predicting the domains for modified peptides in the IMS/MS space that would guide subproteome investigations.
Matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) is a technique that has seen a sharp rise in both use and development. Despite this rapid adoption, there have been few thorough investigations into the actual physical mechanisms that underlie the acquisition of IMS images. We therefore set out to characterize the effect of IMS laser ablation patterns on the surface of a sample. We also concluded that the governing factors that control spatial resolution have not been correctly defined and therefore propose a new definition of resolution.
We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern.
Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R2) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.
Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue.
Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein’s size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds.
A novel concept for ion spatial peak compression is described, and discussed primarily in the context of ion mobility spectrometry (IMS). Using theoretical and numerical methods, the effects of using non-constant (e.g., linearly varying) electric fields on ion distributions (e.g., an ion mobility peak) is evaluated both in the physical and temporal domains. The application of a linearly decreasing electric field in conjunction with conventional drift field arrangements is shown to lead to a reduction in IMS physical peak width. When multiple ion packets (i.e., peaks) in a selected mobility window are simultaneously subjected to such fields, there is ion packet compression (i.e., a reduction in peak widths for all species). This peak compression occurs with only a modest reduction of resolution, which can be quickly recovered as ions drift in a constant field after the compression event. Compression also yields a significant increase in peak intensities. Ion mobility peak compression can be particularly useful for mitigating diffusion-driven peak broadening over very long path length separations (e.g., in cyclic multi-pass arrangements), and for achieving higher S/N and IMS resolution over a selected mobility range.
The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are.
Tissue imaging using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a well-established technique that, in recent years, has seen wider adoption and novel application. Applications such imaging mass spectrometry (IMS) and biotyping are beginning to gain greater exposure and use; however, with limitations in optimization methods, producing the best result often relies on the ability to customize the physical characteristics of the instrumentation, a task that is challenging for most mass spectrometry laboratories. With this in mind, we have described the effect of making simple adjustments to the laser optics at the final collimating lens area, to adjust the laser beam size and shape in order to allow greater customization of the instrument for improving techniques such as IMS. We have therefore been able to demonstrate that improvements can be made without requiring the help of an electrical engineer or external funding in a way that only costs a small amount of time.
Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract.
Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows data-independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors through the modulation of precursor ion cyclotron radii prior to fragmentation. Previous results show that implementation of 2D FT-ICR MS with infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD) has turned this method into a useful analytical tool. In this work, IRMPD tandem mass spectrometry of calmodulin (CaM) has been performed both in one-dimensional and two-dimensional FT-ICR MS using a top-down and bottom-up approach. 2D IRMPD FT-ICR MS is used to achieve extensive inter-residue bond cleavage and assignment for CaM, using its unique features for fragment identification in a less time- and sample-consuming experiment than doing the same thing using sequential MS/MS experiments.
We report relative dephasing cross sections for the 20 biogenic protonated amino acids measured using the cross sectional areas by Fourier transform ion cyclotron resonance (CRAFTI) technique at 1.9 keV in the laboratory reference frame, as well as momentum transfer cross sections for the same ions computed from Boltzmann-weighted structures determined using molecular mechanics. Cross sections generally increase with increasing molecular weight. Cross sections for aliphatic and aromatic protonated amino acids are larger than the average trend, suggesting these side chains do not fold efficiently. Sulfur-containing protonated amino acids have smaller than average cross sections, reflecting the mass of the S atom. Protonated amino acids that can internally hydrogen-bond have smaller than average cross sections, reflecting more extensive folding. The CRAFTI measurements correlate well with results from drift ion mobility (IMS) and traveling wave ion mobility (TWIMS) spectrometric measurements; CRAFTI results correlate with IMS values approximately as well as IMS and TWIMS values from independent measurements correlate with each other. Both CRAFTI and IMS results correlate well with the computed momentum transfer cross sections, suggesting both techniques provide accurate molecular structural information. Absolute values obtained using the various methods differ significantly; in the case of CRAFTI, this may be due to errors in measurements of collision gas pressure, measurement of excitation voltage, and/or dependence of cross sections on kinetic energy.
Dopant-assisted atmospheric pressure chemical ionization (dAPCI) is a soft ionization method rarely used for gas chromatography-mass spectrometry (GC-MS). The current study combines GC-dAPCI with tandem mass spectrometry (MS/MS) for analysis of a complex mixture such as lignin pyrolysis analysis. To identify the structures of volatile lignin pyrolysis products, collision-induced dissociation (CID) MS/MS using a quadrupole time-of-flight mass spectrometer (QTOFMS) and pseudo MS/MS through in-source collision-induced dissociation (ISCID) using a single stage TOFMS are utilized. To overcome the lack of MS/MS database, Compound Structure Identification (CSI):FingerID is used to interpret CID spectra and predict best matched structures from PubChem library. With this approach, a total of 59 compounds were positively identified in comparison to only 22 in NIST database search of GC-EI-MS dataset. This study demonstrates the effectiveness of GC-dAPCI-MS/MS to overcome the limitations of traditional GC-EI-MS analysis when EI-MS database is not sufficient.
Proof of concept evidence is presented for a new method for the determination of isoaspartate, an important post-translational modification. Chemical derivatization is performed using common reagents for the modification of carboxylic acids and shown to yield suitable diagnostic information with regard to isomerization at the aspartate residue. The diagnostic gas phase chemistry is probed by collision-induced dissociation mass spectrometry, on the timescale of the MS experiment and semi-quantitative calibration of the percentage of isoaspartate in a peptide sample is demonstrated.
In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150–250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMS-CID-TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10–200 nM range, while simultaneously achieving discovery measurements of not initially targeted peptides as markers from the same proteins and, eventually, other proteins.
We describe a systematic comparison of high and low resolution LC-MS/MS assays for quantification of 25-hydroxyvitamin D3 in human serum. Identical sample preparation, chromatography separations, electrospray ionization sources, precursor ion selection, and ion activation were used; the two assays differed only in the implemented final mass analyzer stage; viz. high resolution quadrupole-quadrupole-time-of-flight (QqTOF) versus low resolution triple quadrupole instruments. The results were assessed against measured concentration levels from a routine clinical chemiluminescence immunoassay. Isobaric interferences prevented the simple use of TOF-MS spectra for extraction of accurate masses and necessitated the application of collision-induced dissociation on the QqTOF platform. The two mass spectrometry assays provided very similar analytical figures of merit, reflecting the lack of relevant isobaric interferences in the MS/MS domain, and were successfully applied to determine the levels of 25-hydroxyvitamin D for patients with chronic liver disease.
The development of rapid, sensitive, and accurate mass spectrometric methods for measuring peptides, proteins, and even intact protein assemblies has made mass spectrometry (MS) an extraordinarily enabling tool for structural biology. Here, we provide a personal perspective of the increasingly useful role that mass spectrometric techniques are exerting during the elucidation of higher order protein structures. Areas covered in this brief perspective include MS as an enabling tool for the high resolution structural biologist, for compositional analysis of endogenous protein complexes, for stoichiometry determination, as well as for integrated approaches for the structural elucidation of protein complexes. We conclude with a vision for the future role of MS-based techniques in the development of a multi-scale molecular microscope.
