The analysis of many hydrogen exchange (HX) experiments depends on knowledge of exchange rates expected for the unstructured protein under the same conditions. We present here some minor adjustments to previously calibrated values and a stringent test of their accuracy.
Differential or field asymmetric waveform ion mobility spectrometry (FAIMS) operating at high electric fields fully resolves isotopic isomers for a peptide with labeled residues. The naturally present isotopes, alone and together with targeted labels, also cause spectral shifts that approximately add for multiple heavy atoms. Separation qualitatively depends on the gas composition. These findings may enable novel strategies in proteomic and metabolomic analyses using stable isotope labeling.
The flowing atmospheric-pressure afterglow (FAPA) source was used for the mass-spectrometric analysis of vapor samples introduced between the source and mass spectrometer inlet. Through interrupted operation of the plasma-supporting helium flow, helium consumption is greatly reduced and dynamic gas behavior occurs that was characterized by schlieren imaging. Moreover, mass spectra acquired immediately after the onset of helium flow exhibit a signal spike before declining and ultimately reaching a steady level. This initial signal appears to be due to greater interaction of sample vapor with the afterglow of the source when helium flow resumes. In part, the initial spike in signal can be attributed to a pooling of analyte vapor in the absence of helium flow from the source. Time-resolved schlieren imaging of the helium flow during on and off cycles provided insight into gas-flow patterns between the FAPA source and the MS inlet that were correlated with mass-spectral data.
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 present a simple algorithm for robust and unsupervised peak detection by determining a noise threshold in isotopically resolved mass spectrometry data. Solving this problem will greatly reduce the subjective and time-consuming manual picking of mass spectral peaks and so will prove beneficial in many research applications. The Autopiquer approach uses autocorrelation to test for the presence of (isotopic) structure in overlapping windows across the spectrum. Within each window, a noise threshold is optimized to remove the most unstructured data, whilst keeping as much of the (isotopic) structure as possible. This algorithm has been successfully demonstrated for both peak detection and spectral compression on data from many different classes of mass spectrometer and for different sample types, and this approach should also be extendible to other types of data that contain regularly spaced discrete peaks.
Quadrupole mass filters using non-sinusoidal driving potentials present exciting opportunities for new functionality. Predicting figures of merit like resolving power and transmission efficiency helps characterize these emerging devices. To this end, matrix methods of solving the Hill equation of ion motion are employed to calculate stability diagrams and pseudopotential well depth maps in the a,q plane for arbitrary waveforms. The theoretical resolving power and well depth of digital, trapezoidal and sinusoidal mass filters are compared. Simplified expressions for digital mass filter operation are presented.
Matrix recrystallization is optimized and applied to improve lipid ion signals in maize embryos and leaves. A systematic study was performed varying solvent and incubation time. During this study, unexpected side reactions were found when methanol was used as a recrystallization solvent, resulting in the formation of a methyl ester of phosphatidic acid. Using an optimum recrystallization condition with isopropanol, there is no apparent delocalization demonstrated with a transmission electron microscopy (TEM) pattern and maize leaf images obtained at 10 μm spatial resolution.
Precise localization of post-translational modifications (PTMs) on proteins and peptides is an outstanding challenge in proteomics. While electron transfer dissociation (ETD) has dramatically advanced PTM analyses, mixtures of localization variants that commonly coexist in cells often require prior separation. Although differential or field asymmetric waveform ion mobility spectrometry (FAIMS) achieves broad variant resolution, the need for standards to identify the features has limited the utility of approach. Here we demonstrate full a priori characterization of variant mixtures by high-resolution FAIMS coupled to ETD and the procedures to systematically extract the FAIMS spectra for all variants from such data.
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.
A new geometry for the flight region in a time-of-flight mass spectrometer is presented. It consists of two opposing electrostatic sectors of about 255° each and straight sections with a length appropriate to the turns. The resulting geometry folds into a compact space. The first-order aberrations for position, angle, and energy are all zero. The transverse focusing properties are also excellent. For an energetic, high-divergence ion source such as laser ablation, the sTOF has higher resolution and ion transmission than a reflectron of similar physical size.
A modified Kendrick Mass Defect (KMD) analysis was applied to the analysis of polycyclic aromatic hydrocarbons (PAHs) and fullerenes in the diffusion flame from a handheld butane torch.
In negative electrospray ionization mass spectrometry of 4-nitrobenzyl 4-hydroxybenzoates, a decarboxylation reaction, which was significantly promoted by the presence of a nitro group on the benzyl group, competed with radical elimination reactions. Density functional theory calculations indicated that decarboxylation of deprotonated 4-nitrobenzyl vanillate occurred via a radical route involving homolytic cleavage of the Cbenzyl–O bond to give a triplet ion–neutral complex, followed by decarboxylative coupling.
A dual-polarity linear ion trap (LIT) mass spectrometer was developed in this study, and the method for simultaneously controlling and detecting cations and anions was proposed and realized in the LIT. With the application of an additional dipolar DC field on the ejection electrodes of an LIT, dual-polarity mass spectra could be obtained, which include both the mass-to-charge (m/z) ratio and charge polarity information of an ion. Compared with conventional method, the ion ejection and detection efficiency could also be improved by about one-fold. Furthermore, ion–ion reactions within the LIT could be dynamically controlled and monitored by manipulating the distributions of ions with opposite charge polarities. This method was then used to control and study the reaction kinetics of ion–ion reactions, including electron transfer dissociation (ETD) and charge inversion reactions. A dual-polarity collision-induced dissociation (CID) experiment was proposed and performed to enhance the sequence coverage of a peptide ion. Ion trajectory simulations were also carried out for concept validation and system optimization.
One difficult problem in the analysis of peptide modifications is quantifying isomeric modifications that differ by the position of the amino acid modified. HPLC separation using C18 reverse phase chromatography coupled with electron transfer dissociation (ETD) in tandem mass spectrometry has recently been shown to be able to relatively quantify how much of a given modification occurs at each amino acid position for isomeric mixtures; however, the resolution of reverse phase chromatography greatly complicates quantification of isomeric modifications by ETD because of the chromatographic separation of peptides with identical modifications at different sequence positions. Using peptide oxidation as a model system, we investigated the use of size exclusion chromatography coupled with ETD fragmentation to separate peptide sequences. This approach allows for the benefits of chromatographic separation of peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using standard data-dependent acquisitions. Using this method, the relative amount of modification at each amino acid can be accurately measured from single ETD MS/MS spectra in a standard data-dependent acquisition experiment.
Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.
We have developed a multimodal ion source design that can be configured on the fly for various analysis modes, designed for more efficient and reproducible sampling at the mass spectrometer atmospheric pressure (AP) interface in a number of different applications. This vacuum-assisted plasma ionization (VaPI) source features interchangeable transmission mode and laser ablation sampling geometries. Operating in both AC and DC power regimes with similar results, the ion source was optimized for parameters including helium flow rate and gas temperature using transmission mode to analyze volatile standards and drug tablets. Using laser ablation, matrix effects were studied, and the source was used to monitor the products of model prebiotic synthetic reactions.
A new class of compounds, mono- and bis-haloethylphosphonates (HAPs and bisHAPs, respectively), listed in Schedule 2.B.04 of the Chemical Weapons Convention (CWC), has been synthesized and studied by GC-MS with two aims. First, to improve the identification of this type of chemicals by the Organization for the Prohibition of Chemical Weapons, (OPCW). Second, to study the synergistic effect of halogen and silicon atoms in molecules undergoing mass spectrometry. Fragmentation patterns of trimethylsilyl derivatives of HAPs were found to depend on the nature of the halogen atom; this was in agreement with DFT-calculations. The data suggest that a novel intramolecular halogen transfer takes place during the fragmentation process.
The detailed chemical information contained in the vibrational spectrum of a cryogenically cooled analyte ion would, in principle, make infrared (IR) ion spectroscopy a gold standard technique for molecular identification in mass spectrometry. Despite this immense potential, there are considerable challenges in both instrumentation and methodology to overcome before the technique is analytically useful. Here, we discuss the promise of IR ion spectroscopy for small molecule analysis in the context of metabolite identification. Experimental strategies to address sensitivity constraints, poor overall duty cycle, and speed of the experiment are intimately tied to the development of a mass-selective cryogenic trap. Therefore, the most likely avenues for success, in the authors’ opinion, are presented here, alongside alternative approaches and some thoughts on data interpretation.
Dissociation of proteins and peptides by 193 nm ultraviolet photodissociation (UVPD) has gained momentum in proteomic studies because of the diversity of backbone fragments that are produced and subsequent unrivaled sequence coverage obtained by the approach. The pathways that form the basis for the production of particular ion types are not completely understood. In this study, a statistical approach is used to probe hydrogen atom elimination from a + 1 radical ions, and different extents of elimination are found to vary as a function of the identity of the C-terminal residue of the a product ions and the presence or absence of hydrogen bonds to the cleaved residue.
Electrothermal supercharging (ETS) with electrospray ionization produces highly charged protein ions from buffered aqueous solutions in which proteins have native folded structures. ETS increases the charge of ribonuclease A by 34%, whereas only a 6% increase in charge occurs for a reduced-alkylated form of this protein, which is unfolded and its structure is ~66% random coil in this solution. These results indicate that protein denaturation that occurs in the ESI droplets is the primary mechanism for ETS. ETS does not affect the extent of solution-phase hydrogen-deuterium exchange (HDX) that occurs for four proteins that have significantly different structures in solution, consistent with a droplet lifetime that is considerably shorter than observable rates of HDX. Rate constants for HDX of ubiquitin are obtained with a spatial resolution of ~1.3 residues with ETS and electron transfer dissociation of the 10+ charge-state using a single capillary containing a few μL of protein solution in which HDX continuously occurs. HDX protection at individual residues with ETS HDX is similar to that with reagent supercharging HDX and with solution-phase NMR, indicating that the high spray potentials required to induce ETS do not lead to HD scrambling.
