Integrated MICROFASP Method with CZE-Based Fractionation Technique and NanoRPLC-ESI-MS/MS for a Comprehensive Proteomics Analysis of a Submicrogram Sample.

We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 µg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.

An Optimized Miniaturized Filter-Aided Sample Preparation Method for Sensitive Cross-Linking Mass Spectrometry Analysis of Microscale Samples.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for elucidating protein structures and protein-protein interactions (PPIs) at the global scale. However, sensitive XL-MS analysis of mass-limited samples remains challenging, due to serious sample loss during sample preparation of the low-abundance cross-linked peptides. Herein, an optimized miniaturized filter-aided sample preparation (O-MICROFASP) method was presented for sensitive XL-MS analysis of microscale samples. By systematically investigating and optimizing crucial experimental factors, this approach dramatically improves the XL identification of low and submicrogram samples. Compared with the conventional FASP method, more than 7.4 times cross-linked peptides were identified from single-shot analysis of 1 µg DSS cross-linked HeLa cell lysates (440 vs 59). The number of cross-linked peptides identified from 0.5 µg HeLa cell lysates was increased by 58% when further reducing the surface area of the filter to 0.058 mm2 in the microreactor. To deepen the identification coverage of XL-proteome, five different types of cross-linkers were used and each µg of cross-linked HeLa cell lysates was processed by O-MICROFASP integrated with tip-based strong cation exchange (SCX) fractionation. Up to 2741 unique cross-linked peptides were identified from the 5 µg HeLa cell lysates, representing 2579 unique K-K linkages on 1092 proteins. About 96% of intraprotein cross-links were within the maximal distance restraints of 26 Å, and 75% of the identified PPIs reported by the STRING database were with high confidence (scores ≥0.9), confirming the high validity of the identified cross-links for protein structural mapping and PPI analysis. This study demonstrates that O-MICROFASP is a universal and efficient method for proteome-wide XL-MS analysis of microscale samples with high sensitivity and reliability.

Effective microorganism combinations improve the quality of compost-bedded pack products in heifer barns: exploring pack bacteria-fungi interaction mechanisms.

BACKGROUND: Compost-bedded pack barns (CBP) are getting huge attention as an alternative housing system for dairy cows due to their beneficial impact on animal welfare. Effective microorganisms (EM) inoculums are believed to enhance compost quality, improve soil structure and benefit the environment. However, little information is available on the impact of incubation with external EM combinations on the barn environment, compost quality and microbial diversity in CBP. This experiment was carried out to investigate the effect of inoculating different combinations of EM [Lactobacillus plantarum (L), Compound Bacillus (B) and Saccharomyces cerevisiae (S)] on compost quality and microbial communities of CBP products, as well as the relationship with the heifers' barn environment. CBP barns were subjected to the following four treatments: CON with no EM inoculum, LB/LS/LBS were Incubated with weight ratios of 1:2 (L: B), 1:2 (L: S), 1:1:1 (L: B: S), respectively. RESULTS: The EM inoculation (LB, LS, LBS) reduced the concentration of respirable particulate matter (PM10 and PM2.5) in the CBP, and decreased the serum total protein and total cholesterol levels in heifers. Notably, LBS achieved the highest content of high-density lipoprotein compared to other treatments. Microbiome results revealed that EM inoculation reduced the bacterial abundance (Chao1 index) and fungal diversity (Shannon & Simpson indexes), while increasing the relative abundance of various bacterial genera (Pseudomonas, Paracoccus, Aequorivita) and fungi (Pestalotiopsis), which are associated with cellulose decomposition that ultimately resulted in accelerating organic matter degradation and humification. Furthermore, high nutrient elements (TK&TP) and low mycotoxin content were obtained with EM inoculation, with LBS showing a particularly pronounced effect. Meanwhile, LBS contributed to a decline in the proportion of fungal pathogen categories but also led to an increase in fungal saprotroph categories. CONCLUSION: Generally, EM inoculation positively impacted compost product quality as organic fertilizer and barn environment by modifying the abundance of cellulolytic bacteria and fungi, while inhibiting the reproduction of pathogenic microbes, especially co-supplementing with L, B and S achieved an amplifying effect.

Topological corner state localized bound states in continuum in photonic crystals.

In the field of optics, bound states in the continuum (BICs) are of significant practical importance as they can trap electromagnetic waves spatially, even though their frequency lies within the continuous spectrum. Previous research, however, has shown that BICs localized in optical cavities are highly sensitive to geometric and environmental changes. This sensitivity implies that slight variations can lead to the loss of BICs, necessitating extreme precision in manufacturing, which poses a challenge for practical implementation. To overcome this issue, this study employs topological photonic crystals (PhCs) to engineer topological corner states (TCS) within PhCs. By doing so, it establishes a method for creating topological BICs that are inherently robust against disturbances, thereby enhancing their suitability for real-world applications.

Capillary zone electrophoresis-tandem mass spectrometry for in-depth proteomics analysis via data-independent acquisition.

A capillary zone electrophoresis (CZE) system was coupled to an Orbitrap mass spectrometer operating in a data-independent acquisition (DIA) mode for in-depth proteomics analysis. The performance of this CZE-DIA-MS system was systemically evaluated and optimized under different operating conditions. The performance of the fully optimized CZE-DIA-MS system was subsequently compared to the one by using the same CZE-MS system operating in a data-dependent acquisition (DDA) mode. The experimental results show that the numbers of identified peptides and proteins acquired in the DIA mode are much higher than the ones acquired in the DDA mode, especially with the small sample loading amount. Specifically, the numbers of identified peptides and proteins acquired in the DIA mode are 1.8-fold and 2-fold higher than the ones acquired in the DDA mode by using 12.5 ng Hela digests. The proteins identified in the DIA mode also cover almost all the proteins identified in the DDA mode. In addition, a potential cancer biomarker protein, carbohydrate antigen 125, undetected in the DDA mode, can be easily identified in the DIA mode even with 12.5 ng Hela digests. The performance of the CZE-DIA-MS system for in-depth proteomics analysis with a limited sample amount has been fully demonstrated for the first time through this study.

A Facile and High-Sensitivity Method for Determining Proteinogenic Amino Acid Enantiomers by Integrating Chiral Phosphinate Derivatizing, 31P NMR and Parallel Reaction Monitoring.

Here, we have documented a new protocol to determine d/l-amino acids by derivatizing amino acids via a chiral phosphinate. (RP)-l-Menthyl phenylphosphinate was able to bond both primary and secondary amines, as well as improve the sensitivity of analytes in MS. Eighteen pairs of amino acids were successfully labeled except for Cys which has a thiol group on the side chain, and the chirality of amino acids can be discriminated by 31P NMR. Seventeen pairs of amino acids were separated by a C18 column within 45 min of elution, and resolution values ranged from 2.01 to 10.76. The lowest limit of detection was 10 pM acquired at parallel reaction monitoring, in which two factors collectively contributed that the ability of protonation of phosphine oxide and the sensitivity of parallel reaction monitoring. Chiral phosphine oxides might be a promising tool in future chiral metabolomics.

Combining sensitivity and robustness: EIT-like characteristic in a 2D topological photonic crystal.

The study of topological photonics has gained significant attention due to its potential application for robust and efficient light manipulation. In this work, we theoretically investigate a two-dimensional photonics crystal that exhibits a topological edge state (TES) and a topological corner state (TCS). Furthermore, we also achieve a coupling between a topological corner state and a trivial cavity (TC), resulting in a phenomenon similar to the electromagnetically induced transparency (EIT) effect. To verify the stability of the EIT-like effect, disorders around TES and TCS are introduced, and the theoretical results show that this structure is immune to the disorders. The achievement of the coupling between topological states can have potential applications in the areas of waveguiding, sensing, and logic gates. It is hoped that this work will contribute to the ongoing efforts in the exploration and utilization of topological photonics.

The value of serum neutralizing antibody in evaluating predictability of COVID-19 after recovery and the validation of vaccine.

BACKGROUND: This work aimed to study natural humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Chemiluminescent immunoassay (CLIA) was used to detect the neutralizing antibody (Nabs) and IgG. RESULTS: Nabs peaked on days 57-96 after symptom onset and remained detected on days 97-132. The Nabs in the 32 patients who were dynamically monitored showed four changing patterns. The titers of Nabs and IgG were correlated, and three modes of relationship were found between them. CONCLUSIONS: Nabs showed a regular change in the course of coronavirus disease 2019 (COVID-19). The detection of Nabs is very important for monitoring the course of COVID-19 and predicting the strength of antibody protection.

Slow-light effects based on the tunable Fano resonance in a Tamm state coupled graphene surface plasmon system.

We theoretically realize the tunable Fano resonance in a hybrid structure that allows the coupling between Tamm plasmon-polaritons (TPPs) and graphene surface plasmon-polaritons (SPPs). In this coupling system, a distributed Bragg reflector (DBR)/Ag structure is designed to generate the TPP with a narrow resonance, and the graphene SPP is excited by grating coupling with a broad resonance. The overlap of these two kinds of resonances results in the Fano resonance with a high-quality factor close to 1500. The behaviors of the Fano resonance are discussed carefully, and the results show that both the graphene Fermi level and the incidence angle can actively tune the profile of the Fano resonance. Owing to the ultrasharp spectrum of the tunable Fano resonance, our design may offer an alternative strategy for developing various optoelectronic devices such as filters, sensors, and nonlinear and slow-light devices. Finally, as an example of the potential applications, we apply the tunable Fano resonance to the slow-light effect, a high performance slow-light effect can be achieved, and the group delay can reach up to 52 ps.

Generation of symmetry-protected bound states in the continuum in a graphene plasmonic waveguide system for optical switching.

A graphene-involved plasmonic lossy system that allows coupling between surface plasmon polaritons and waveguide modes is proposed. The physical mechanism behind the hybrid resonance modes is investigated carefully through finite element method (FEM) simulations and rigorous coupled wave theory (RCWA). We demonstrate that by introducing an incident angle to break the symmetry of the structure, the bound states in the continuum (BIC) evolve to an observable quasi-BIC with new resonance dips, and the generated signals possess a very high Q-factor. Such transformation is investigated carefully by calculating the band structure of the system and the corresponding Q-factors. The results showed that the calculated results from the band structure are consistent with the simulations. In addition, the hybrid plasmonic system allows for switching modulation due to the tunability of graphene, and the max modulation depth of nearly 100% is reached. The outstanding Q-factor and dynamic tunability of this easy-to-fabricate hybrid structure may be helpful in engineering various plasmonic devices, including tunable optical switches, absorbers, sensors, etc.

Seamlessly Integrated Miniaturized Filter-Aided Sample Preparation Method to Fractionation Techniques for Fast, Loss-Less, and In-Depth Proteomics Analysis of 1 µg of Cell Lysates at Low Cost.

We report an integrated platform that enabled a seamlessly coupling miniaturized filter-aided sample preparation (MICROFASP) method to high-pH reversed phase (RP) or strong cation exchange (SCX) microreactors for low-loss sample preparation and fractionation of 1 µg of cell lysates prior to LC-ESI-MS/MS analysis. Due to the reduced size of the microreactor, only 5 µL of buffer volume is required to generate each fraction, which speeds both elution and lyophilization. The fraction was directly eluted into an autosampler insert vial for LC-MS analysis to reduce sample transfer steps and minimize sample loss as well as contamination. The flow-through sample generated during the loading step was also collected and analyzed. The integrated platform generated 48,890 unique peptides and 4723 protein groups from 1 µg of a K562 cell lysate using MICROFASP and C18 microreactor-based high-pH RP fractionation methods, which are comparable with the state-of-the-art result using in-StageTip sample preparation and nanoflow RPLC-based fractionation methods but with a significant reduction in cost and time. Both pH gradient elution and salt gradient elution approaches provide high reproducibility for the SCX microreactor-based fractionation method. This integrated platform has significant potential in deep proteomics analysis of mass-limited samples with reduced time and equipment requirements.

High-Throughput, Comprehensive Single-Cell Proteomic Analysis of Xenopus laevis Embryos at the 50-Cell Stage Using a Microplate-Based MICROFASP System.

We report both the design of a high-throughput MICROFASP (a miniaturized filter aided sample preparation) system and its use for the comprehensive proteomic analysis of single blastomeres isolated from 50-cell stage Xenopus laevis embryos (∼200 ng of yolk-free protein/blastomere). A single run of the MICROFASP system was used to process 146 of these blastomeres in parallel. Three samples failed to generate signals presumably due to membrane clogging. Two cells were lost due to operator error. Of the surviving samples, 32 were analyzed using a Q Exactive HF mass spectrometer in survey experiments (data not included). The 109 remaining blastomeres were analyzed using a capillary LC-ESI-MS/MS system coupled to an Orbitrap Fusion Lumos mass spectrometer, which identified a total of 4189 protein groups and 40,998 unique peptides. On average, 3468 ± 229 protein groups and 14,525 ± 2437 unique peptides were identified from each blastomere, which is the highest throughput and deepest proteome coverage to date of single blastomeres at this stage of development. We also compared two dissociation buffers, Newport and calcium-magnesium-free (CMFM) buffers; the two buffers generated similar numbers of protein identifications (3615 total protein IDs from use of the Newport dissociation buffer and 3671 total protein IDs from use of the CMFM buffer).

Hydrophilic, dual amino acid-functionalized zinc sulfide quantum dot for specific identification of N-glycopeptides from biological samples.

RATIONALE: Glycosylation of proteins is one of the most significant and complex post-translational modifications, and N-glycosylation plays a crucial role in life activities. Mass spectrometry (MS) has been a powerful technique in the analysis of protein glycosylation. However, the direct detection of glycoproteins in biological samples based on MS still suffers from huge challenges. Therefore, enrichment and purification of samples before MS analysis is an essential prerequisite. METHODS: Hydrophilic interaction liquid chromatography (HILIC) has significantly developed for selective enrichment of glycopeptides due to its simple operation process and unbiased enrichment. Herein, hydrophilic, dual amino acid-functionalized zinc sulfide quantum dots (ZnS QDs) were prepared to enrich glycopeptides using an easy procedure. The enriched glycopeptides were detected using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: The obtained material exhibited high selectivity (1:2000), low detection limit (0.1 fmol/µl), good repeatability (10 times), and excellent recovery (89.8%) in glycopeptide enrichment. In the actual application in biological samples, 71 N-glycopeptides and 161 N-glycopeptides were detected from human saliva and serum, respectively. CONCLUSIONS: ZnS-Au-GC was successfully prepared using an easy method. The results showed that the obtained material exhibited excellent performance in glycopeptide enrichment. Furthermore, it had showed great potential for glycopeptide enrichment in complex biological samples.

Dynamically tunable bound states in the continuum supported by asymmetric Fabry-Pérot resonance.

The dynamic regulation of quasi-bound states in the continuum (quasi-BIC) is a research hotspot, such as incident angle, polarization angle, temperature, a medium refractive index, and medium position regulation. In this paper, a dual-band ultra-high absorber composed of upper asymmetric graphene strips and lower graphene nanoribbons can generate a symmetry-protected quasi-BIC and Fabry-Pérot resonance (FPR) mode. The band structure further demonstrates the symmetry-protected BIC. Research shows that the absorption system can withstand a relatively wide range of incidence and polarization angles. Interestingly, the quasi-BIC and FPR modes can be modulated by the Fermi levels of the graphene1 and graphene2, respectively, realizing a multifunctional switch with high modulation depth (MD > 94%), low insertion loss (IL < 0.23 dB), and large dephasing time (DT > 4.35 ps). This work provides a new approach for the dynamic regulation of quasi-BIC and stimulates the development of multifunctional switches in the absorber.

Triple plasmon-induced transparency and optical switch desensitized to polarized light based on a mono-layer metamaterial.

A mono-layer metamaterial comprising four graphene-strips and one graphene-square-ring is proposed herein to realize triple plasmon-induced transparency (PIT). Theoretical results based on the coupled mode theory (CMT) are in agreement with the simulation results obtained using the finite-difference time-domain (FDTD). An optical switch is investigated based on the characteristics of graphene dynamic modulation, with modulation degrees of the amplitude of 90.1%, 80.1%, 94.5%, and 84.7% corresponding to 1.905 THz, 2.455 THz, 3.131 THz, and 4.923 THz, respectively. Moreover, the proposed metamaterial is insensitive to the change in the angle of polarized light, for which the triple-PIT is equivalent in the cases of both x- and y-polarized light. The optical switch based on the proposed structure is effective not only for the linearly polarized light in different directions but also for left circularly polarized and right circularly polarized light. As such, this work provides insight into the design of optoelectronic devices based on the polarization characteristics of the incident light field on the optical switch and PIT.

Quadruple plasmon-induced transparency of polarization desensitization caused by the Boltzmann function.

This study proposes a graphene metamaterial desensitized to the polarized angle to produce tunable quadruple plasmon-induced transparency (PIT). As a tool employed to explain the PIT, n-order coupled mode theory (CMT) is deduced for the first time and closely agrees with finite-difference time-domain (FDTD) simulations according to the quadruple PIT results in the case of n = 5. Additionally, the response of the proposed structure to the angle of polarized light is investigated. As a result, the Boltzmann function satisfied by the response of graphene strips to the polarization direction of incident light is proposed for the first time. Its property of polarization desensitization can be attributed to structural centrosymmetry, and conjugated variety which the Boltzmann functions result in. Therefore, a quintuple-mode modulation based on simultaneous electro-optical switch is realized by tuning Fermi levels within graphene. Its modulation degrees of amplitude and dephasing times are obtained. Given that the slow-light property is an important application of PIT, the n-order group index is thereby obtained. Hence, not only do the insights gained into polarization-desensitization structure provide new ideas for the design of novel optoelectronic devices, but also the results from the n-order CMT offer new research progress and references in theory.

Broadband plasmon-induced transparency modulator in the terahertz band based on multilayer graphene metamaterials.

In this study, multilayer graphene metamaterials comprising graphene blocks and graphene ribbon are proposed to realize dynamic plasmon-induced transparence (PIT). By changing the position between the graphene blocks, PIT phenomenon will occur in different terahertz bands. Furthermore, PIT with a transparent window width of 1 THz has been realized. In addition, the PIT shows redshifts or blueshifts or disappears altogether upon changing the Fermi level of graphene, and hence a frequency selector from 3.91 to 7.84 THz and an electro-optical switch can be realized. Surprisingly, the group index of this structure can be increased to 469. Compared with the complex and fixed structure of previous studies, our proposed structure is simple and can be dynamically adjusted according to demands, which makes it a valuable platform for ideas to inspire the design of novel electro-optic devices.

Miniaturized Filter-Aided Sample Preparation (MICRO-FASP) Method for High Throughput, Ultrasensitive Proteomics Sample Preparation Reveals Proteome Asymmetry in Xenopus laevis Embryos.

We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm2 surface area, reduces the total volume of reagents to <10 µL, and requires only two sample transfer steps. The method was used to generate 25 883 unique peptides and 3069 protein groups from 1000 MCF-7 cells (∼100 ng protein content), and 13 367 peptides and 1895 protein groups were identified from 100 MCF-7 cells (∼10 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere) generated 20 943 unique peptides and 2597 protein groups; the proteomic profile clearly differentiated left and right blastomeres and provides strong support for models in which this asymmetry is established early in the embryo. The parallel processing of 12 samples demonstrates reproducible label free quantitation of 1 µg protein homogenates.

Polarization-sensitive triple plasmon-induced transparency with synchronous and asynchronous switching based on monolayer graphene metamaterials.

A monolayer graphene metamaterial comprising four graphene strips and four graphene blocks is proposed to produce triple plasmon-induced transparency (PIT) by the interaction of three bright modes and one dark mode. The response of the proposed structure is analyzed by using couple mode theory and finite-difference time-domain simulations, with the results of each method showing close agreement. A quadruple-mode on-to-off modulation based on synchronous or asynchronous switching is realized by tuning the Fermi levels in the graphene, its modulation degrees of amplitude are 77.7%, 58.9%, 75.4%, and 77.6% corresponding to 2.059 THz, 2.865 THz, 3.381 THz, and 3.878 THz, respectively. Moreover, the influence of the polarized light angle on triple-PIT is investigated in detail, demonstrating that the polarization angle affects PIT significantly. As a result, a multi-frequency polarizer is realized, its polarization extinction ratios are 4.2 dB, 7.8 dB, and 12.5 dB. Combined, the insights gained into the synchronous or asynchronous switching and the polarization sensitivity of triple-PIT provide a valuable platform and ideas to inspire the design of novel optoelectronic devices.

Photoelectric switch and triple-mode frequency modulator based on dual-PIT in the multilayer patterned graphene metamaterial.

A multilayer patterned graphene metamaterial composed of rectangular graphene, square graphene, and X-shaped graphene is proposed to achieve dual plasmon-induced transparency (PIT) at terahertz frequency. The coupled mode theory calculations are highly consistent with the finite-difference time-domain numerical results. Interestingly, a photoelectric switch has been realized, whose extinction ratio and modulation degree of amplitude can be 7.77 dB and 83.3% with the insertion loss of 7.2%. In addition, any dips can be modulated by tuning the Fermi levels of three graphene layers with minor or ignorable changes of the other two dips. The modulation degrees of frequency are 8.0%, 7.4% and 11.7%, respectively, which can be used to design a triple-mode frequency modulator. Moreover, the group index of the multilayer structure can be as high as 150. Therefore, it is reasonable to believe that a multifunctional device can be realized by the proposed structure.