Gas-phase Separation Research Articles

Utilization of Fast Photochemical Oxidation of Proteins and Both Bottom-up and Top-down Mass Spectrometry for Structural Characterization of a Transcription Factor-dsDNA Complex.

A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein-DNA complex. Data are available via ProteomeXchange with identifier PXD027624.

Free-Standing Nanometer-Thick Covalent Organic Framework Films for Separating CO2 and N2

Covalent organic frameworks (COF) have been gathering much attention because the shapes, sizes, and chemical functions of their nanostructured pores can be arbitrarily controlled by designing the organic precursors. We fabricated cm-sized free-standing COF films with the thickness of 50–100 nm by an alternating vacuum deposition polymerization method. Precise adjustment of the stoichiometry by digitally controlled deposition was essential for producing the robust free-standing COF films. High-resolution electron microscopy revealed 3 nm pore structures, which correspond to the atomic structure of the COF. Small angle X-ray diffraction shows the existence of a similar periodicity. The CO2/N2 gas phase separation properties were evaluated from 103–105 Pa, and the parameters for each molecular permeation were determined. Based on the detailed analysis, it was found that the selectivity comes from the greater sorption affinity of CO2 to the COF compared to N2, which is consistent with the quantum chemical calculation. Since the vapor phase method can be used to coat various shaped templates, our method provides a new option for fabrication of neat COF membranes with various structures and their applications for the separation membrane.

A Miniature Multilevel Structures for Lossless Ion Manipulations Ion Mobility Spectrometer with Wide Mobility Range Separation Capabilities.

Ion mobility spectrometry employing structures for lossless ion manipulations (SLIM-IMS) is an attractive gas-phase separation technique due to its ability to achieve unprecedented effective ion path lengths (>1 km) and IMS resolving powers in a small footprint. The emergence of multilevel SLIM technology, where ions are transferred between vertically stacked SLIM electrode surfaces, has subsequently allowed for ultralong single-pass path lengths (>40 m) to be achieved, enabling ultrahigh resolution IMS measurements to be performed over the entire mobility range in a single experiment. Here, we report on the development of a 1 m path length miniature SLIM module (miniSLIM) based on multilevel SLIM technology. Ion trajectory simulations were used to optimize SLIM board spacings and SLIM board thicknesses, and a new method of efficiently transferring ions between SLIM levels using asymmetric traveling waves (TWs) was demonstrated. We experimentally characterized the performance of the miniSLIM IMS-MS relative to a drift tube IMS-MS using Agilent tuning mixture cations and tetraalkylammonium cations. The miniSLIM achieved a resolving power of up to 131 (CCS/ΔCCS), which is ∼1.5× higher than achievable with a 78 cm path length drift tube IMS. Additionally, the entire ion mobility range was successfully transmitted in a single separation. We also demonstrated the miniSLIM's performance as a standalone IMS system (i.e., without MS), which showed baseline separation between all AgTM cations and a clear differentiation between different charge states of a standard peptide mixture. Overall, the miniSLIM provides a compact alternative to high performance IMS instruments possessing similar path lengths.

Separation of motions and vibrational separation of fractions for biocide brass

The mathematical method of separation of motions represents the effect of fast high-frequency oscillations by an effective averaged force or potential. Ultrasound acoustic vibrations are an example of such rapid oscillations leading to cavitation in water due to the gas phase formation (bubbles). Ultrasound cavitation is used to treat the surface of brass microparticles submerged in water. The formation of bubbles and their collapse triggers the modification of surface roughness and chemical composition. Consequently, the suspension separates into various fractions related to demonstrating biocide properties. While the exact mechanism of this process is complex, it can be explained phenomenologically by using the Onsager reciprocal relations for coupling the copper ion diffusion with the gas phase separation in water as a result of the action of the effective average vibrational force.

Identification and Mitigation of Defensins in the Immunopurification of Peptide MHC-I Antigens from Lung Tissue.

The class I major histocompatibility (MHC-I) complex is a set of diverse cell surface receptors encoded by the human leukocyte antigen gene complex. These receptors present intracellular antigens to cytotoxic T cells providing information on the state and health of cells. Changes in the immunopeptidome during cancer may provide novel targets for therapeutic intervention. To understand how the tumor immunopeptidome is altered, we developed a mass spectrometry (MS) based platform for isolating and identifying MHC-I peptide antigens in lung tumors. In the course of our work, we encountered several large unknown peptide contaminants which had not been previously reported. To understand the source of these major contaminants, we isolated them using offline fractionation and identified them by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as members of the host defense protein family known as the defensins. To mitigate their detrimental effects, we modified our "Original" data-dependent acquisition (DDA) MS method to narrowly target the MHC-I peptides based on their physical properties including charge state and molecular weight ("z state" DDA), evaluated field asymmetric ion mobility spectrometry to attempt gas-phase separation prior to MS analysis, and developed an immunodepletion approach using defensin specific antibodies. This modified approach improves peptide identification and reduces the impact of defensin contamination in lung tissue samples.

Numerical investigation on spilling upward performance of hydrogen bubbles inside a delivery tube under low-gravity environment

A typical cryogenic upper stage mission requires a reliable liquid–gas phase separation prior to engine restart in space, and auxiliary engines are usually used to provide positive acceleration effect to satisfy vapor-free liquid supplement purpose. To assist thruster logic design for an upper stage, bubble distribution inside a delivery tube and bubble spilling behaviors should be known previously. In the present paper, a CFD approach is adopted to numerically study the bubble generation and bubble movement in liquid hydrogen (LH2) delivery tube under microgravity, and the bubble spilling upward behaviors under different situations are significantly analyzed. The results show that space heat from surroundings brings about liquid evaporation inside the delivery tube, and weak convection effect induced by bubble generation process could result in random bubble distribution in the whole tube range. The maximum bubble reaches the size of tube inner diameter. Under positive acceleration effect, the bubbles spill upward and bring about gas amount decrease in the delivery tube range. Moreover, the bubble spilling upward is not a continuous process, and only the big bubble discharge exerts a significant contribution to the gas amount decrease. In addition, the tube layout and the initial gas amount have slight influence on the bubble discharge performance, but the space acceleration level plays the primary effect on this process. For the present objective, the acceleration with 10−3g0 could drive the bubble to be discharged within 700 s. When the acceleration increases to 10−1g0, the required time correspondingly decreases to less than 100 s. Under the acceleration level of 10−4g0, however, the bubble could not spill upward any more but a vapor amount increase phenomenon is observed. Generally, the present work provides an opportunity to understand the bubble properties inside the cryogenic delivery tube in microgravity, and the results on acceleration influence as well as acceleration period are beneficial to the sequence design of space engine restart.

Investigations into pesticide charge site isomers using conventional IM and cIM systems

A growing number of pesticides are being used around the world necessitating strict regulatory policies to guarantee consumer safety. Liquid Chromatography – Mass Spectrometry (LC-MS) is a highly sensitive method for pesticide screening, which provides retention time, mass/charge ratios and the relative abundances of characteristic product ions. Variability in the latter necessitates relatively large tolerances (±30%, SANCO/12682/2019, current EU regulation). One cause of this variability may stem from the presence of different charge-site isomers (charge carrier being a proton, sodium cation, potassium cation and alike); each yielding a set of different product ions, of which the relative ratios are influenced by solution and ion source conditions. Consequently, varying relative abundances may be observed for analyte ions produced from calibration standards, chemical residues in food matrices and across different instruments. Ion Mobility Spectrometry (IMS) is a fast, gas phase separation technique which can resolve charge-site isomers based on differences in their collisional cross sections (CCSs). We previously used the IM device embedded in LC-IM-MS geometry to generate a pesticide CCS database and subsequently focussed upon identification of pesticides which form charge-site isomers. Latterly, we applied this approach to screen food commodities for pesticide residues. In some instances, isomer separation was clear, however sometimes broad, unresolved distributions were observed. Using a high-resolution cyclic IM device (cIM) we resolved and determined CCS values of species of indoxacarb, spinosad, fenpyroximate, epoxiconazole, metaflumizone and avermectin. Furthermore, utilising novel cIM functionalities (tandem-IM) we discovered that two spinosyn sodimers can interconvert in the gas phase.

A Compendium of Murine (Phospho)Peptides Encompassing Different Isobaric Labeling and Data Acquisition Strategies.

Targeted mass spectrometry-based assays typically rely on previously acquired large data sets for peptide target selection. Such repositories are widely available for unlabeled peptides. However, they are less common for isobaric tagged peptides. Here we have assembled two series of six data sets originating from a mouse embryonic fibroblast cell line (NIH/3T3). One series is of peptides derived from a tryptic digest of a whole cell proteome and a second from enriched phosphopeptides. These data sets encompass three labeling approaches (unlabeled, TMT11-labeled, and TMTpro16-labeled) and two data acquisition strategies (ion trap MS2 with and without FAIMS-based gas phase separation). We identified a total of 1 509 526 peptide-spectrum matches which covered 11 482 proteins from the whole cell proteome tryptic digest, and 188 849 phosphopeptides from the phosphopeptide enrichment. The data sets were of similar depth, and while overlap across data sets was modest, protein overlap was high, thus reinforcing the comprehensiveness of these data sets. The data also supported FAIMS as a means to increase data set depth. These data sets provide a rich resource of peptides that may be used as starting points for targeted assays. Future data sets may be compiled for any genome-sequenced organism using the technologies and strategies highlighted herein. The data have been deposited in the ProteomeXchange Consortium with data set identifier PXD024298.

Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down Proteomics.

Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.

Comprehensive Screening of Polycyclic Aromatic Hydrocarbons and Similar Compounds Using GC-APLI-TIMS-TOFMS/GC-EI-MS.

In the present work, a novel workflow based on complementary gas-phase separations for the identification of isomeric PAHs from complex mixtures is described. This is the first report on the coupling of gas chromatography (GC), atmospheric pressure laser ionization (APLI), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) for the characterization of polycyclic aromatic hydrocarbons. Over a hundred known unknowns are uniquely identified based on the molecular ion retention indices I (5%), mobility (RSD < 0.6% and R = 50-90 with Sr = 0.18 V/ms), mobility-based theoretical candidate assignment (<3%), accurate mass chemical formula assignment (<2 ppm), and electron impact fragmentation pattern and database search. The advantages of theoretical modeling of PAHs and similar compounds were evaluated using candidate structures ranked by retention indices and fragmentation pattern from GC-EI-MS data sets. Over 20 PAH isomeric and deuterated standards were utilized for the GC-APLI-TIMS-TOF MS workflow validation. Noteworthy is the analytical capability for untargeted screening of isomeric and isobaric compounds with additional characterization metrics not available in traditional GC-EI-MSn workflows.

Bench Tests and CFD Simulations of Liquid–Gas Phase Separation Modeling with Simultaneous Liquid Transport and Mechanical Foam Destruction

This paper presents simulation and bench test results for a special type of centrifugal pump that enables the transport of dispersive foaming liquids and simultaneous separation of the liquid phase. During the design phase, CFD (Computer Fluid Dynamics) simulations were performed using Ansys Fluent. The simulations covered changing the operating parameters of the pump (mass/volume flow rate), pressure analysis for the first impeller, and structural optimization of the pump components. In the second stage of the research, the pump and a measuring station were constructed to validate the results of the numerical simulations.

Effect of two-step calcination on the formation of nickel oxide hollow nanofibers

1D (one-dimensional) hollow nanofibers are of great technological interest. Their formation can be controlled by tuning the calcination process. However, the formation mechanism through the control of the burning stage is still debated in the literature. Herein, the influence of calcination in two steps on the morphology of hollow nanofibers of nickel oxide (NiO), prepared by solution blow spinning (SBS) technique, was investigated. A systematic study using a variety of characterization techniques suggests that a prolonged residence time in the initial step of calcination produces nanofibers with a better definition of the hollow region with better control of tubular wall thickness. Statistically, the calcination methodology studied here does not affect the outer diameter of nanofibers, as shown by Analysis of Variance (ANOVA) and Tukey’s method. According to our findings, the formation mechanism is influenced by gas diffusion and phase separation during fiber spinning and heat treatment, which suggests that neither the Kirkendall effect nor the Ostwald ripening process can be solely responsible for the formation hollow fibers. These findings contribute to a better understanding of the formation of metal-oxide hollow nanofibers induced by heat treatment.

Reevaluating the Role of Polarizability in Ion Mobility Spectrometry

With the expanding commercial availability of gas-phase separation systems that incorporate gas-phase mobility, there is a concurrent rise in efforts to cast the gas-phase mobility coefficient in terms of an ion-neutral collision cross-section (CCS). The motivating factors for this trend are varied, but many aim to complement experimental results with computationally generated CCS values from in silico structural approximations. Unfortunately, the current paradigm for relating experimental mobility results to computationally derived structures relies upon empirical approaches, including a myriad of variables that do not realistically bound the comparison. In this Critical Insight, we advocate for the development of a self-consistent experimental and computational framework that uses laboratory results to constrain the scope of the modeling effort. This paper aims to prompt discussion, challenge assumptions, and promote the development of more efficient, accurate computational techniques within the gas-phase ion measurement community. Specifically, we postulate whether experimental deviations from Langevin's polarization limit (Kpol) are suitable to estimate the relative contributions of hard-sphere collisions and long-range interactions within CCS values. Not surprisingly, different molecule classes exhibit different trends in the K/Kpol ratio when normalized for reduced mass, and the most common IMS calibrants (e.g., tune mix, polyalanine, tetraalkylammonium salts) follow different polarizability trends than many of the analytes probed in the literature. Succinctly, if gas-phase ion structure is largely invariant based upon the colliding neutral and newly developed experimental efforts can quantitatively capture ion polarizability, then modeling efforts describing a target analyte must be self-consistent as the collision neutral is changed in silico.

BoxCarmax: A High-Selectivity Data-Independent Acquisition Mass Spectrometry Method for the Analysis of Protein Turnover and Complex Samples.

The data-independent acquisition (DIA) performed in the latest high-resolution, high-speed mass spectrometers offers a powerful analytical tool for biological investigations. The DIA mass spectrometry (DIA-MS) combined with the isotopic labeling approach holds a particular promise for increasing the multiplexity of DIA-MS analysis, which could assist the relative protein quantification and the proteome-wide turnover profiling. However, the wide MS1 isolation windows employed in conventional DIA methods lead to a limited efficiency in identifying and quantifying isotope-labeled peptide pairs through peptide fragment ions. Here, we optimized a high-selectivity DIA-MS named BoxCarmax that supports the analysis of complex samples, such as those generated from Stable isotope labeling by amino acids in cell culture (SILAC) and pulse SILAC (pSILAC) experiments. BoxCarmax enables multiplexed acquisition at both MS1 and MS2 levels, through the integration of BoxCar and MSX features, as well as a gas-phase separation strategy. We found BoxCarmax significantly improved the quantitative accuracy in SILAC and pSILAC samples by mitigating the ratio suppression of isotope-peptide pairs. We further applied BoxCarmax to measure protein degradation regulation during serum starvation stress in cultured cells, revealing valuable biological insights. Our study offered an alternative and accurate approach for the MS analysis of protein turnover and complex samples.

Simulation study on phase separation and pressure distribution of refrigerant in horizontal double T-junctions

T-junction is widely used as a phase separation component to separate two-phase flow because of its simple structure and low cost. Previous studies suggest that an increase in the number of T-junction branches is conducive to raise phase separation efficiency. In this paper, the pressure distribution and phase separation performance of two-phase flow of R134a in horizontal double T-junctions are studied. The phase separation performance between two branches is compared. The parameters including inlet quality (0.3-0.6), inlet mass flow rate (10.0 g s−1-25.0 g s−1), diameter ratio (1, 0.75) and the distance between two branches (0.3 m-2.6 m) are studied. The inlet mass flow rate has significant influence on the phase separation, and the separation performance reaches best when it is 10.0 g s−1. Results show that the change of parameters has greater influence on the phase separation performance of the second branch compared with the first branch. When the distance between branches is 2.0 m, the total phase separation capacity of the branches reaches the best. By comparing the phase separation of branches, it can be found that when the distance increases gradually, the phase separation performance of the second branch first increases and then decreases, indicating that there is a specific distance that maximizes the gas phase separation efficiency of branches. The optimal distance is also related to the mass flow ratio of the second branch. Therefore, an empirical correlation equation which can accurately predict the gas phase separation efficiency of the second branch is proposed.

Different ion mobility-mass spectrometry coupling techniques to promote metabolomics.

Metabolomics has become increasingly popular in recent years for many applications ranging from clinical diagnosis, human health to biotechnological questioning. Despite technological advances, metabolomic studies are still currently limited by the difficulty of identifying all metabolites, a class of compounds with great chemical diversity. Although lengthy chromatographic analyses are often used to obtain comprehensive data, many isobar and isomer metabolites still remain unresolved, which is a critical point for the compound identification. Currently, ion mobility spectrometry is being explored in metabolomics as a way to improve metabolome coverage, analysis throughput and isomer separation. In this review, all the steps of a typical workflow for untargeted metabolomics are discussed considering the use of an ion mobility instrument. An overview of metabolomics is first presented followed by a brief description of ion mobility instrumentation. The ion mobility potential for complex mixture analysis is discussed regarding its coupling with a mass spectrometer alone, providing gas-phase separation before mass analysis as well as its combination with different separation platforms (conventional hyphenation but also multidimensional ion mobility couplings), offering multidimensional separation. Various instrumental and analytical conditions for improving the ion mobility separation are also described. Finally, data mining, including software packages and visualization approaches, as well as the construction of ion mobility databases for the metabolite identification are examined.

High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry.

Ion mobility (IM) is a gas phase separation strategy that can either supplement or serve as a high-throughput alternative to liquid chromatography (LC) in shotgun lipidomics. Incorporating the IM dimension in untargeted lipidomics workflows can help resolve isomeric lipids, and the collision cross section (CCS) values obtained from the IM measurements can provide an additional molecular descriptor to increase lipid identification confidence. This chapter provides a broad overview of an untargeted ion mobility-mass spectrometry (IM-MS) workflow using a commercial drift tube ion mobility-quadrupole-time-of-flight mass spectrometer (IM-QTOF) for high confidence lipidomics.

Pore size characterization of micro-mesoporous carbons using CO2 adsorption

Pore structure characterization plays a crucial role in the optimization of adsorption properties of nanoporous carbons employed for water purification, gas and liquid phase separations, carbon dioxide reduction, energy storage, and other applications. Here, we present an original methodology for evaluating the pore size distribution in carbons in a wide range of micro- and mesopores from 0.385 to 10 nm from a single isotherm of high-pressure adsorption of CO2 at 273 K. The proposed method is based on the reference theoretical isotherms calculated by Monte Carlo simulations in model pores of slit-shaped and cylindrical geometry. The relationship between the pore size and the pore filling pressure is established. Special attention is given to the predicting of the capillary condensation transitions in mesopores by using the meso-canonical ensemble (gauge cell) Monte Carlo simulations. The proposed technique is demonstrated and verified against the conventional N2 and Ar low temperature adsorption methods drawing on the example of micro-mesoporous carbons of the CMK family. Advantages and limitations of CO2 adsorption characterization of nanoporous materials are discussed and further improvements are proposed.

Phase separation and secondary migration of methane-rich gas accompanying uplift of an unconventional tight-hydrocarbon system, Montney Formation, western Canada

Tight-hydrocarbon systems can undergo significant depressurization through leakage of fluids, primarily gas, in response to uplift. Leakage at the up-dip margins of these systems may be highly variable if internal gas transmission by secondary migration is focused along fairways with favourable stratigraphic and structural characteristics. In this study of the Montney Formation, regional mapping of the methane content of produced gas reveals two orders of trends. The first-order regional trend is increasing methane content with depth and thermal maturity. Second-order trends are orthogonal or oblique to the first-order trend, and defined by curvilinear fairways with methane contents higher than expected from the first-order trend. They can be mapped for 10s km with some extending up-dip from the dry gas window into the oil window. Methane enrichment along second-order trends reflects fractionation of hydrocarbon fluids by a variety of processes including phase separation, secondary migration, fluid mixing and evaporative fractionation. Gas composition, recombination PVT and carbon isotope ratio data from produced fluids are used in three local case studies to show that methane-rich gas was driven up-dip by buoyancy forces and regional pressure gradient with migration focused along suitably oriented fairways of enhanced permeability and positive local structure. Secondary migration of methane-rich gas resulted in modified hydrocarbon compositions with leaner liquid contents and higher gas-oil ratios than expected from thermal maturity indicators. Recognition of secondary methane migration fairways is thus essential for hydrocarbon evaluation and exploitation in the Montney Formation, and conceivably in other analogous tight-hydrocarbon systems that have undergone significant uplift.

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.