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.
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.
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.
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.
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.
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.
Characterization of the cysteine content of proteins is a key aspect of proteomics. By defining both the total number of cysteines and their bound/unbound state, the number of candidate proteins considered in database searches is significantly constrained. Herein we present a methodology that utilizes 266 nm UVPD to count the number of free and bound cysteines in intact proteins. In order to attain this goal, proteins were derivatized with N-(phenylseleno)phthalimide (NPSP) to install a selectively cleavable Se–S bond upon 266 UVPD. The number of Se–S bonds cleaved upon UVPD, a process that releases SePh moieties, corresponds to the number of cysteine residues per protein.
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.
A number of proteomic database search engines implement multi-stage strategies aiming at increasing the sensitivity of proteome analysis. These approaches often employ a subset of the original database for the secondary stage of analysis. However, if target-decoy approach (TDA) is used for false discovery rate (FDR) estimation, the multi-stage strategies may violate the underlying assumption of TDA that false matches are distributed uniformly across the target and decoy databases. This violation occurs if the numbers of target and decoy proteins selected for the second search are not equal. Here, we propose a method of decoy database generation based on the previously reported decoy fusion strategy. This method allows unbiased TDA-based FDR estimation in multi-stage searches and can be easily integrated into existing workflows utilizing popular search engines and post-search algorithms.
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.
A high performance liquid chromatograph (HPLC)was interfaced to an atmospheric drift tube ion mobility time of flight mass spectrometry. The power of multidimensional separation was demonstrated using chili pepper extracts. The ambient pressure drift tube ion mobility provided high resolving powers up to 166 for the HPLC eluent. With implementation of Hadamard transform (HT), the duty cycle for the ion mobility drift tube was increased from less than 1% to 50%, and the ion transmission efficiency was improved by over 200 times compared with pulsed mode, improving signal to noise ratio 10 times. HT ion mobility and TOF mass spectrometry provide an additional dimension of separation for complex samples without increasing the analysis time compared with conventional HPLC.
A collision induced dissociation (CID) structure for lossless ion manipulations (SLIM) module is introduced and coupled to a quadrupole time-of-flight (QTOF) mass spectrometer. The SLIM CID module was mounted after an ion mobility (IM) drift tube to enable IM/CID/MS studies. The efficiency of CID was studied by using the model peptide leucine enkephalin. CID efficiencies (62%) compared favorably with other beam-type CID methods. Additionally, the SLIM CID module was used to fragment a mixture of nine peptides after IM separation. This work also represents the first application of SLIM in the 0.3 to 0.5 Torr pressure regime, an order of magnitude lower in pressure than previously studied.
Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin–drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.
Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein’s function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications.
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.
The utility of sodium ion adducts produced by matrix-assisted laser desorption ionization for the quantification of analytes with multiple oxygen atoms was evaluated. Uses of homogeneous solid samples and temperature control allowed the acquisition of reproducible spectra. The method resulted in a direct proportionality between the ion abundance ratio I([A?+?Na]+)/I([M?+?Na]+) and the analyte concentration, which could be used as a calibration curve. This was demonstrated for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude.
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 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.
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.
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.
