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.
Ion mobility mass spectrometry (IM-MS) holds great potential for structural glycobiology, in particular in its ability to resolve glycan isomers. Generally, IM-MS has largely been applied to intact glycoconjugate ions with reports focusing on the separation of different adduct types. Here, we explore IM separation and report the collision cross section (CCS) of complex type N-glycans and their fragments in negative ion mode following collision-induced dissociation (CID). CCSs of isomeric fragment ions were found, in some cases, to reveal structural details that were not present in CID spectra themselves. Many fragment ions were confirmed as possessing multiple structure, details of which could be obtained by comparing their drift time profiles to different glycans. By using fragmentation both before and after mobility separation, information was gathered on the fragmentation pathways producing some of the ions. These results help demonstrate the utility of IM and will contribute to the growing use of IM-MS for glycomics.
A cationic degradation product, formed in solution from retinal Schiff base (RSB), is examined in the gas phase using ion mobility spectrometry, photoisomerization action spectroscopy, and collision induced dissociation (CID). The degradation product is found to be N-n-butyl-2-(β-ionylidene)-4-methylpyridinium (BIP) produced through 6π electrocyclization of RSB followed by protonation and loss of dihydrogen. Ion mobility measurements show that BIP exists as trans and cis isomers that can be interconverted through buffer gas collisions and by exposure to light, with a maximum response at λ = 420 nm.
Ion mobility-mass spectrometry (IM-MS) has recently seen increased use in the analysis of small molecules, especially in the field of metabolomics, for increased breadth of information and improved separation of isomers. In this study, steroid epimers androsterone and trans-androsterone were analyzed with IM-MS to investigate differences in their relative mobilities. Although sodiated monomers exhibited very similar collision cross-sections (CCS), baseline separation was observed for the sodiated dimer species (RS = 1.81), with measured CCS of 242.6 and 256.3 Å2, respectively. Theoretical modeling was performed to determine the most energetically stable structures of solution-phase and gas-phase monomer and dimer structures. It was revealed that these epimers differ in their preferred dimer binding mode in solution phase: androsterone adopts a R=O – Na+ – OH—R′ configuration, whereas trans-androsterone adopts a R=O – Na+ – O=R′ configuration. This difference contributes to a significant structural variation, and subsequent CCS calculations based on these structures relaxed in the gas phase were in agreement with experimentally measured values (ΔCCS ~ 5%). Additionally, these calculations accurately predicted the relative difference in mobility between the epimers. This study illustrates the power of combining experimental and theoretical results to better elucidate gas-phase structures.
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.
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 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.
Structural isomers and enantiomers of hexachlorocyclohexane (HCH) were separated using a chiral column by gas chromatography and quantitatively determined by multiphoton ionization mass spectrometry using an ultraviolet femtosecond laser (200 and 267 nm) as the ionization source. The order of elution of the enantiomers (i.e., (+)-α-HCH and (-)-α-HCH) was predicted from stabilization energies calculated for the complexes using permethylated γ-cyclodextrin as the stationary phase of the column, and the results were compared with the experimental data. The molecular ions observed for HCH were weak, even though they can be ionized through a process of resonance enhanced two-photon ionization at 200 nm. This unfavorable result can be attributed to the dissociation of the molecular ion, as predicted from quantum chemical calculations.
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.
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.
First results are reported using a simple, fast, and reproducible matrix-assisted ionization (MAI) sample introduction method that provides substantial improvements relative to previously published MAI methods. The sensitivity of the new MAI methods, which requires no laser, high voltage, or nebulizing gas, is comparable to those reported for MALDI-TOF and n-ESI. High resolution full acquisition mass spectra having low chemical background are acquired from low nanoliters of solution using only a few femtomoles of analyte. The limit-of-detection for angiotensin II is less than 50 amol on an Orbitrap Exactive mass spectrometer. Analysis of peptides, including a bovine serum albumin digest, and drugs, including drugs in urine without a purification step, are reported using a 1 μL zero dead volume syringe in which only the analyte solution wetting the walls of the syringe needle is used in the analysis.
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.
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.
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.
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 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.
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.
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.
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.
