Asymmetric Waveform Research Articles

Studying Structural Details in Complex Samples: II. High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Coupled to High Resolution Tandem Mass Spectrometry (MS/MS).

The elucidation of structural motifs in extremely complex mixtures is very difficult since the standard methods for structural elucidation are not capable to provide significant information on a single molecule. The best method for the analysis of complex mixtures is ultrahigh resolution mass spectrometry, but the utilization of this method alone does not provide significant information about structural details. Here, a combination with a separation method is necessary. While chromatography is a well-established technique, it has some disadvantages in regard to the separation of complex mixtures, as often no separation of individual isomers is possible. Therefore, here the combination of an ion mobility separation with ultrahigh resolution mass spectrometry is evaluated. As a sample matrix, crude oil is used because it is an excellent matrix to develop new analytical techniques on complex samples. Crude oil is the most complex natural sample known, but only little information is available on the structural identity or functionalities due to a high number of structural isomers or isobars. A lab-built APPI/APLI-FAIMS source was revised to optimize ion transmission and used to follow up on the ion mobility of crude oil constituents after photoionization. An MS/MS approach using collision-induced dissociation (CID) was used to elucidate structural motifs of the transmitted isomers.

The fascicular anatomy of the sciatic nerve may not influence the muscular activity of the biceps femoris after applying percutaneous electrical nerve stimulation: a randomized clinical trial.

Electrical nerve stimulation is an effective therapeutic tool in the field of rehabilitation for the management of musculoskeletal conditions. To analyze the muscle activity of the short head of the biceps femoris (SHBF) and long head of the biceps femoris (LHBF) after the application of percutaneous electrical nerve stimulation to the common fibular versus tibial nerve compartment of the sciatic nerve. Thirty-eight healthy subjects received electrical stimulation (asymmetric biphasic rectangular waveforms) through a needle in the sciatic nerve. The sample was divided into two groups in which the technique was applied to the lateral compartment versus medial compartment of the nerve, respectively. The protocol used included 5 s of stimulation at a frequency of 100 Hz and a phase duration of 350 ms, followed by 55 s of rest, for 5 min. The electrical activity of the SHBF and LHBF and the muscle strength of the knee flexors was assessed by surface electromyography during isometric knee flexion in the prone position. Neither group showed significant changes (p > 0.05) in any variable after the intervention compared with baseline values. Similarly, there were no significant differences in any of the study variables when comparing both groups (p > 0.05). Selective stimulation of the sciatic nerve by percutaneous electrical nerve stimulation had no measurable effects on muscle activity in the SHBF or LHBF, regardless of the approach applied. NCT05549700 (ClinicalTrials.gov).

N-terminomics profiling of naïve and inflamed murine colon reveals proteolytic signatures of legumain.

Legumain is a cysteine protease broadly associated with inflammation. It has been reported to cleave and activate protease-activated receptor 2 to provoke pain associated with oral cancer. Outside of gastric and colon cancer, little has been reported on the roles of legumain within the gastrointestinal tract. Using a legumain-selective activity-based probe, LE28, we report that legumain is activated within colonocytes and macrophages of the murine colon, and that it is upregulated in models of acute experimental colitis. We demonstrated that loss of legumain activity in colonocytes, either through pharmacological inhibition or gene deletion, had no impact on epithelial permeability in vitro. Moreover, legumain inhibition or deletion had no obvious impacts on symptoms or histological features associated with dextran sulfate sodium-induced colitis, suggesting its proteolytic activity is dispensable for colitis initiation. To gain insight into potential functions of legumain within the colon, we performed field asymmetric waveform ion mobility spectrometry-facilitated quantitative proteomics and N-terminomics analyses on naïve and inflamed colon tissue from wild-type and legumain-deficient mice. We identified 16 altered cleavage sites with an asparaginyl endopeptidase signature that may be direct substrates of legumain and a further 16 cleavage sites that may be indirectly mediated by legumain. We also analyzed changes in protein abundance and proteolytic events broadly associated with colitis in the gut, which permitted comparison to recent analyses on mucosal biopsies from patients with inflammatory bowel disease. Collectively, these results shed light on potential functions of legumain and highlight its potential roles in the transition from inflammation to colorectal cancer.

Development of LC-FAIMS-MS and its application to lipidomics study of Acinetobacter baumannii infection

The recent advances in mass spectrometry (MS) technologies have enabled comprehensive lipid profiling in biological samples. However, the robustness and efficiency of MS-based lipidomics is compromised by the complexity of biological samples. High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a technology that can continuously transmit one type of ion, independent of the mass-to-charge ratio. Here we present the development and application of LC-FAIMS-MS/MS-based platform for untargeted lipidomics. We used 3 optimally balanced compensation voltages, i.e., 29 V, 34 V and 39 V, to analyze all subclasses of glycerophospholipids. The reproducibility of the method was evaluated using reference standards. The reproducibility of retention times ranged from 0.9% to 1.5% RSD; whereas RSD values of 5%–10% were observed for peak areas. More importantly, the coupling of a FAIMS device can significantly improve the robustness and efficiency. We exploited this NPLC-FAIMS-HRMS to analyze the serum lipid profiles in mice infected intranasally with Acinetobacter baumannii. The temporal profiles of serum lipids after A. baumannii inoculation were obtained for 4 h, 8 h, and 24 h. We found that nearly all ether PC and ether PE lipids were significantly decreased 8 h after inoculation. The resultant volcano plot illustrated the distribution of 28 increased and 28 decreased lipid species in mouse sera 24 h after inoculation. We also found that a single ether PE composition can comprise multiple isomeric structures, and the relative abundance of each isomer could be quantified using the newly developed NPLC-FAIMS-PRM method. We have demonstrated that the proposed LC-FAIMS-MS is a valuable platform for lipidomics.

Preparation and optical characterization of 3D-TiO2 thin films with brilliant colors

In this paper, three-dimensional titanium dioxide (3D-TiO2) thin films with periodic structure were obtained by periodic pulse anodization of high-purity titanium wafers under specific alternating voltage (AV) using asymmetric sawtooth waveforms. The effect of voltage amplitude and electrolyte concentration on the structural color of the 3D-TiO2 thin films were investigated. It is shown that the nanotubes grown by asymmetric sawtooth wave at a specific AV have a “bamboo” shape, and the cycle length, as well as the inner and outer tube diameters of the nanotubes are closely related to the AV. The structural color of the samples was different at varying voltages and electrolyte concentrations for the same oxidation cycle time. The structural color of films grown under low voltage is analyzed to be related to the scattering and absorption of light on their surfaces. The thickness of the film grown under high voltage satisfies the conditions for light interference. The maximum reflected wavelength is red-shifted and then blue-shifted as the electrolyte concentration decreases. The study shows that the microstructure is the main influence on the color change of 3D-TiO2 thin films.

Racetrack FAIMS for High-Resolution and High-Sensitivity Characterization of Peptide Conformers.

A racetrack field asymmetric waveform ion mobility spectrometry (r-FAIMS) device, which consists of both cylindrical FAIMS (c-FAIMS) and planar FAIMS (p-FAIMS) sections with a 1 mm gap width, was developed and applied for high-resolution and high-sensitivity exploration of conformational diversity for peptides. The optimal operating conditions of r-FAIMS were systemically studied, and the performance of the fully optimized r-FAIMS was compared to a previously developed p-FAIMS in detail by using pure nitrogen as the FAIMS carrier gas. Relying on the ion focusing effect in the c-FAIMS section, the intensity of the FAIMS spectrum for doubly charged bradykinin ions acquired by using r-FAIMS is ∼8.5-fold higher than that acquired by using p-FAIMS under the same resolving power/resolution condition, implying about an order of magnitude better sensitivity of r-FAIMS. In addition, the peak separation resolution of r-FAIMS was ∼1.70-fold higher than p-FAIMS under a similar sensitivity condition for doubly charged bradykinin ions. Due to a reduced gap width of the newly designed r-FAIMS (1 mm) as compared to the previously developed p-FAIMS (1.88 mm), r-FAIMS can operate at a much higher separation field with a similar FAIMS dispersion voltage (DV) to gain significantly higher resolving power. For triply charged syntide 2 ions, the resolving power of r-FAIMS can easily exceed 120 at -3.5 kV DV by using pure nitrogen as the FAIMS carrier gas as compared to 44.2 resolving power obtained by using p-FAIMS at -4.0 kV DV. All of the experimental results have confirmed that r-FAIMS can perform structural characterization of biomolecules with both high resolution and high sensitivity.

Hybrid simulation of a capacitive Ar/SiH4 discharge driven by electrically asymmetric voltage waveforms

Voltage waveforms associated with the electrical asymmetry effect (EAE) have the potential to be used in the deposition of the silicon-based film, since they are expected to decouple ion energy and flux at the wafer surface, and further facilitate control of the process. In this study, a one-dimensional fluid/electron Monte Carlo hybrid model is employed to examine the EAE in a capacitively coupled argon-silane discharge, encompassing both amplitude asymmetry effect (AAE) and slope asymmetry effect (SAE). In the case of AAE, with the increasing pressure, the discharge electronegativity gradually intensifies, in conjunction with a transition of the electron heating mode from α to drift-ambipolar, a reduction of the absolute value of the DC self-bias voltage, and a decrease in Ar+ content, with an increase in SiH3 + content. For SAE, the trend in the discharge characteristics with the increasing pressure is similar to that for AAE, but the details are different. In SAE, the electronegativity and bulk electric field are much enhanced, resulting in higher content of high-energy electrons and Ar+ in the bulk. In addition, the absolute value of the self-bias is lower, but shows a fewer decline with the increasing pressure. The deposition rate is lower in SAE, due to the lower electron heating efficiency. However, larger voltage drop difference between two sheaths leads to a wider range of ion energy modulation at higher pressures. This study systematically investigates and compares Ar/SiH4 discharges driven by two electrically asymmetric voltage waveforms across various parameters including electron dynamics, ion and neutral transport properties, and deposition rates, with the aim of providing valuable insights and a reference for industrial applications.

Effect of dynamic exclusion and the use of FAIMS, DIA and MALDI-mass spectrometry imaging with ion mobility on amyloid protein identification

Amyloidosis is a disease characterized by local and systemic extracellular deposition of amyloid protein fibrils where its excessive accumulation in tissues and resistance to degradation can lead to organ failure. Diagnosis is challenging because of approximately 36 different amyloid protein subtypes. Imaging methods like immunohistochemistry and the use of Congo red staining of amyloid proteins for laser capture microdissection combined with liquid chromatography tandem mass spectrometry (LMD/LC–MS/MS) are two diagnostic methods currently used depending on the expertise of the pathology laboratory. Here, we demonstrate a streamlined in situ amyloid peptide spatial mapping by Matrix Assisted Laser Desorption Ionization–Mass Spectrometry Imaging (MALDI-MSI) combined with Trapped Ion Mobility Spectrometry for potential transthyretin (ATTR) amyloidosis subtyping. While we utilized the standard LMD/LC–MS/MS workflow for amyloid subtyping of 31 specimens from different organs, we also evaluated the potential introduction in the MS workflow variations in data acquisition parameters like dynamic exclusion, or testing Data Dependent Acquisition combined with High-Field Asymmetric Waveform Ion Mobility Spectrometry (DDA FAIMS) versus Data Independent Acquisition (DIA) for enhanced amyloid protein identification at shorter acquisition times. We also demonstrate the use of Mascot’s Error Tolerant Search and PEAKS de novo sequencing for the sequence variant analysis of amyloidosis specimens.

Improved breast milk proteome coverage by DIA based LC-MS/MS method.

The breast milk composition includes a multitude of bioactive factors such as viable cells, lipids and proteins. Measuring the levels of specific proteins in breast milk plasma can be challenging because of the large dynamic range of protein concentrations and the presence of interfering substances. Therefore, most proteomic studies of breast milk have been able to identify under 1000 proteins. Optimised procedures and the latest separation technologies used in milk proteome research could lead to more precise knowledge of breast milk proteome. This study (n=53) utilizes three different protein quantification methods, including direct DIA, library-based DIA method and a hybrid method combining direct DIA and library-based DIA. On average we identified 2400 proteins by hybrid method. By applying these methods, we quantified body mass index (BMI) associated variation in breast milk proteomes. There were 210 significantly different proteins when comparing the breast milk proteome of obese and overweight mothers. In addition, we analysed a small cohort (n=5, randomly selected from 53 samples) by high field asymmetric waveform ion mobility spectrometry (FAIMS). FAIMS coupled with the Orbitrap Fusion Lumos mass spectrometer, which led to 41.7% higher number of protein identifications compared to Q Exactive HF mass spectrometer.

Improvement of Tumor Neoantigen Detection by High-Field Asymmetric Waveform Ion Mobility Mass Spectrometry.

Cancer neoantigens have been shown to elicit cancer-specific T-cell responses and have garnered much attention for their roles in both spontaneous and therapeutically induced antitumor responses. Mass spectrometry (MS) profiling of tumor immunopeptidomes has been used, in part, to identify MHC-bound mutant neoantigen ligands. However, under standard conditions, MS-based detection of such rare but clinically relevant neoantigens is relatively insensitive, requiring 300 million cells or more. Here, to quantitatively define the minimum detectable amounts of therapeutically relevant MHC-I and MHC-II neoantigen peptides, we analyzed different dilutions of immunopeptidomes isolated from the well-characterized T3 mouse methylcholanthrene (MCA)-induced cell line by MS. Using either data-dependent acquisition or parallel reaction monitoring (PRM), we established the minimum amount of material required to detect the major T3 neoantigens in the presence or absence of high field asymmetric waveform ion mobility spectrometry (FAIMS). This analysis yielded a 14-fold enhancement of sensitivity in detecting the major T3 MHC-I neoantigen (mLama4) with FAIMS-PRM compared with PRM without FAIMS, allowing ex vivo detection of this neoantigen from an individual 100 mg T3 tumor. These findings were then extended to two other independent MCA-sarcoma lines (1956 and F244). This study demonstrates that FAIMS substantially increases the sensitivity of MS-based characterization of validated neoantigens from tumors.

Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques

The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.

Data-Independent Acquisition: A Milestone and Prospect in Clinical Mass Spectrometry–Based Proteomics

Data-independent acquisition (DIA) has revolutionized the field of mass spectrometry (MS)-based proteomics over the past few years. DIA stands out for its ability to systematically sample all peptides in a given m/z range, allowing an unbiased acquisition of proteomics data. This greatly mitigates the issue of missing values and significantly enhances quantitative accuracy, precision, and reproducibility compared to many traditional methods. This review focuses on the critical role of DIA analysis software tools, primarily focusing on their capabilities and the challenges they address in proteomic research. Advances in MS technology, such as trapped ion mobility spectrometry, or high field asymmetric waveform ion mobility spectrometry require sophisticated analysis software capable of handling the increased data complexity and exploiting the full potential of DIA. We identify and critically evaluate leading software tools in the DIA landscape, discussing their unique features, and the reliability of their quantitative and qualitative outputs. We present the biological and clinical relevance of DIA-MS and discuss crucial publications that paved the way for in-depth proteomic characterization in patient-derived specimens. Furthermore, we provide a perspective on emerging trends in clinical applications and present upcoming challenges including standardization and certification of MS-based acquisition strategies in molecular diagnostics. While we emphasize the need for continuous development of software tools to keep pace with evolving technologies, we advise researchers against uncritically accepting the results from DIA software tools. Each tool may have its own biases, and some may not be as sensitive or reliable as others. Our overarching recommendation for both researchers and clinicians is to employ multiple DIA analysis tools, utilizing orthogonal analysis approaches to enhance the robustness and reliability of their findings.

Combining FAIMS based glycoproteomics and DIA proteomics reveals widespread proteome alterations in response to glycosylation occupancy changes in Neisseria gonorrhoeae.

Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of Neisseria gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

Periodic asymmetric function waveform generator based on polarization multiplexing modulator

Periodic asymmetric function waveform generator based on polarization multiplexing modulator

Highly sensitive and selective sulfur hexafluoride detection based on DAPPI-FAIMS

This paper proposed a novel detection method of sulfur hexafluoride (SF6) by combining dopant-assisted atmospheric pressure photoionization (DAPPI) with high-field asymmetric waveform ion mobility spectroscopy (FAIMS). Acetone was employed as a dopant to achieve high sensitivity, and the negative ion mode of FAIMS was selected to achieve high selectivity. The impact of dopant concentrations on detection sensitivity and linearity was studied, and the influence of relative humidity on the detection of sulfur hexafluoride was studied. Ammonia, benzene, and hydrogen sulfide were selected as interferences to verify the selectivity. A linearity between the concentration and the signal was studied, and the detection limit for sulfur hexafluoride was determined. Experimental results demonstrated that the DAPPI source effectively enhanced the ionization efficiency of sulfur hexafluoride with a linear coefficient of 96.1 %. The sensitivity of sulfur hexafluoride is inversely proportional to relative humidity. Sulfur hexafluoride has a characteristic fingerprint spectrum different from air background and typical interfering, and can be distinguished from interfering substances in mixtures containing ammonia, benzene, and hydrogen sulfide. The concentration of sulfur hexafluoride has excellent linearity with signal intensity, the linear coefficient was 99.8 %, and the detection limit was 0.02 ppm.

A pilot clinical assessment of biphasic asymmetric pulsed field ablation catheter for pulmonary vein isolation.

https://www.chictr.org.cn/, identifier, ChiCTR2100051894.

Design and experiment of high-field asymmetric waveform ion mobility spectrometry chip based on an integrated temperature control printed circuit board structure.

During the detection of volatile organic compounds (VOC) using high-field asymmetric waveform ion mobility spectrometry (FAIMS), the ambient temperature significantly impacts the accuracy of planar FAIMS. To mitigate the influence of ambient temperature on detection accuracy and enhance resolution, a FAIMS system based on the inner impedance characteristics of a printed circuit board (PCB) was designed for temperature control. This study, conducted under standard atmospheric pressure, aimed to assess the signal stability of a planar FAIMS instrument with and without temperature control, and the effect of temperature change on the detection ability of acetone, ethanol, and their mixture was studied using PCB self-heating. Experimental results demonstrated that the base noise in FAIMS with temperature control was 0.2pA, whereas that in FAIMS without temperature control was 1.8pA. Notably, with increasing temperature, the detection ability of FAIMS changes accordingly. The optimal relative detection ability of acetone was observed when the electrode plate was heated to 45°C under an electric field of 15 kV/cm. This study provides a novel approach to improve the resolving power of FAIMS systems and their signal-to-noise ratio. The utilization of a PCB-based temperature control proved effective in stabilizing FAIMS signal characteristics and optimizing detection capabilities, particularly for VOCs such as acetone. These findings have significant implications for improving the accuracy and resolving power of FAIMS systems in VOC detection applications.

Ion Mobility–Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of FAIMS as an accessible method to improve depth and quality of N-terminomics studies.

A core loss estimation method based on improved waveform coefficient Steinmetz equation for asymmetric triangular flux density waveform

Accurate estimation of high frequency magnetic core losses under non-sinusoidal excitations is critical to the design of solid-state transformers. This paper proposes a waveform transformation principle, termed the equal derivative transformation (EDT), for piecewise linear flux density waveforms. Under the same maximum flux density, an asymmetric triangular flux density wave can be transformed into a symmetric one with a different frequency in terms of the calculation of core losses, hence the introduction of the improved waveform coefficient Steinmetz equation (iWcSE). The magnetic characteristics of three different toroidal samples (nanocrystalline alloy FT-3KL, amorphous alloy 1K101, ferrite N87) under square excitation at different duty ratios and certain frequencies are measured. Core losses predicted by the model are in good agreement with the measurement.

Swimming ability and flagellar motility of sperm packets of the volvocine green alga Pleodorina starrii.

Eukaryotic flagella collectively form metachronal waves that facilitate the ability to cause flow or swim. Among such flagellated and planktonic swimmers, large volvocine genera such as Eudorina, Pleodorina and Volvox form bundles of small male gametes (sperm) called "sperm packets" for sexual reproduction. Although these sperm packets reportedly have flagella and the ability to swim, previous studies on volvocine motility have focused on asexual forms and the swimming characteristics of sperm packets remain unknown. However, it is important to quantify the motility of sperm packets and sperm in order to gain insights into the significance of motility in the sexual reproduction of planktonic algae. In this study, we quantitatively described the behavior of three flagellated forms of a male strain of Pleodorina starrii-asexual colonies, sperm packets, and single dissociated sperm-with emphasis on comparison of the two multicellular forms. Despite being smaller, sperm packets swam approximately 1.4 times faster than the asexual colonies of the same male strain. Body length was approximately 0.5 times smaller in the sperm packets than in asexual colonies. The flagella from sperm packets and asexual colonies showed asymmetric waveforms, whereas those from dissociated single sperm showed symmetric waveforms, suggesting the presence of a switching mechanism between sperm packets and dissociated sperm. Flagella from sperm packets were approximately 0.5 times shorter and had a beat period approximately twice as long as those from asexual colonies. The flagella of sperm packets were densely distributed over the anterior part of the body, whereas the flagella of asexual colonies were sparse and evenly distributed. The distribution of flagella, but not the number of flagella, appear to illustrate a significant difference in the speeds of sperm packets and asexual colonies. Our findings reveal novel aspects of the regulation of eukaryotic flagella and shed light on the role of flagellar motility in sexual reproduction of planktonic algae.