Fully Unattended Online Protein Digestion and LC-MS Peptide Mapping.

LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC-MS peptide mapping. The method generates LC-MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC-MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC-MS analysis.

Method for detecting rare differences between two LC-MS runs.

LC-MS based multi-attribute methods (MAM) have drawn substantial attention due to their capability of simultaneously monitoring a large number of quality attributes of a biopharmaceutical product. For successful implementation of MAM, it is usually considered a requirement that the method is capable of detecting any new or missing peaks in the sample when compared to a control. Comparing a sample to a control for rare differences is also commonly practiced in many fields for investigational purpose. Because MS signal variability differs greatly between signals of different intensities, this type of comparison is often challenging, especially when the comparison is made without enough replicates. In this report we describe a statistical method for detecting rare differences between two very similar samples without replicate analyses. The method assumes that an overwhelming majority of components have equivalent abundance between the two samples, and signals with similar intensities have similar relative variability. By analyzing several monoclonal antibody peptide mapping datasets, we demonstrated that the method is suitable for new-peak detection for MAM as well as for other applications when rare differences between two samples need to be detected. The method greatly reduced false positive rate without a significant increase of false negative rate.

Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

MULTI-parametric MR imaging with fLEXible design (MULTIPLEX).

PURPOSE: To introduce a gradient echo (GRE) -based method, namely MULTIPLEX, for single-scan 3D multi-parametric MRI with high resolution, signal-to-noise ratio (SNR), accuracy, efficiency, and acquisition flexibility. THEORY: With a comprehensive design with dual-repetition time (TR), dual flip angle (FA), multi-echo, and optional flow modulation features, the MULTIPLEX signals contain information on radiofrequency (RF) B1t fields, proton density, T1 , susceptibility and blood flows, facilitating multiple qualitative images and parametric maps. METHODS: MULTIPLEX was evaluated on system phantom and human brains, via visual inspection for image contrasts and quality or quantitative evaluation via simulation, phantom scans and literature comparison. Region-of-interest (ROI) analysis was performed on parametric maps of the system phantom and brain scans, extracting the mean and SD of the T1 , T2∗ , proton density (PD), and/or quantitative susceptibility mapping (QSM) values for comparison with reference values or literature. RESULTS: One MULTIPLEX scan offers multiple sets of images, including but not limited to: composited PDW/T1 W/ T2∗ W, aT1 W, SWI, MRA (optional), B1t map, T1 map, T2∗ / R2∗ maps, PD map, and QSM. The quantitative error of phantom T1 , T2∗ and PD mapping were <5%, and those in brain scans were in good agreement with literature. MULTIPLEX scan times for high resolution (0.68 × 0.68 × 2 mm3 ) whole brain coverage were about 7.5 min, while processing times were <1 min. With flow modulation, additional MRA images can be obtained without affecting the quality or accuracy of other images. CONCLUSION: The proposed MUTLIPLEX method possesses great potential for multi-parametric MR imaging.

Augmented T1 -weighted steady state magnetic resonance imaging.

T1 contrasts obtained using short-TR incoherent steady state gradient echo (GRE) methods are generally suboptimal, to which non-T1 factors in the signals play a major part. In this work, we proposed an augmented T1 -weighted (aT1 W) method to extract the signal ratio between routine GRE T1 W and proton density-weighted signals that effectively removes the non-T1 effects from the original T1 W signals, including proton density, T2 * decay, and coil sensitivity. A recently proposed multidimensional integration (MDI) technique was incorporated in the aT1 W calculation for better signal-to-noise ratio (SNR) performance. For comparison between aT1 W and T1 W results, Monte Carlo noise analysis was performed via simulation and on scanned data, and region-of-interest (ROI) analysis and comparison was performed on the system phantom. For brain scans, the image contrast, noise behavior, and SNR of aT1 W images were compared with routine GRE and inversion-recovery-based T1 W images. The proposed aT1 W method yielded saliently improved T1 contrasts (potentially > 30% higher contrast-to-noise ratio [CNR]) than routine GRE T1 W images. Good spatial homogeneity and signal consistency as well as high SNR/CNR were achieved in aT1 W images using the MDI technique. For contrast-enhanced (CE) imaging, aT1 W offered stronger post-CE contrast and better boundary delineation than T1 MPRAGE images while using a shorter scan time.

The effectiveness of esketamine and propofol versus dezocine and propofol sedation during gastroscopy: A randomized controlled study.

WHAT IS KNOWN AND OBJECTIVE: Propofol is widely used in painless gastroscopy. However, sedation with propofol alone might increase the risk of respiratory and circulatory complications. This randomized clinical study compares the efficacy and safety of esketamine or dezocine combined with intravenous (IV) propofol in patients undergoing gastroscopy. METHODS: A total of 102 patients were enrolled in this study and randomized into two groups. All patients were adults aged 18-64 years who underwent upper gastrointestinal gastroscopy. Patients were randomly assigned to two groups to receive esketamine (0.3 mg/kg) combined with propofol (group E) or dezocine (0.05 mg/kg) combined with propofol (group D). In both groups, the drugs were administered intravenously. The primary outcome was the dose of propofol which provided a satisfactory sedative effect, both to the endoscopist and the patients. Secondary outcomes included recovery time, side effects (such as hypotension, nausea and vomiting and agitation), and the number of adverse circulatory and respiratory events. RESULTS: Data of 83 patients were analysed in the present study. Dosage of propofol required in group E (1.44 mg/kg ± 0.67 mg/kg) was significantly lower compared with that in group D (2.12 mg/kg ± 0.37 mg/kg) (p < 0.0001). There was no statistically significant difference in recovery time, side effects, and the frequency of sedation-related adverse events between the two groups. WHAT IS NEW AND CONCLUSION: The study indicates that intravenous injection of propofol and esmketamine is more effective for gastroscopy. Use of esketamine reduces the total amount of propofol required in ASA I-II patients undergoing gastroscopy compared with single use of dezocine. It also provides more stable hemodynamics, without affecting the recovery time and side effects such as respiratory and circulatory adverse events. TRIAL REGISTRATION: The study was registered at the Chinese Clinical Trial Registry (www.chictr.org.cn; registration number: ChiCTR2100051814) on 05/10/2021.

Limited Proteolysis Coupled with Mass Spectrometry for Simultaneous Evaluation of a Large Number of Protein Variants for Their Impact on Conformational Stability.

A stable molecular structure is important in the development of a protein candidate into a therapeutic product. A therapeutic protein often contains many different variants; some of them may have an impact on the conformational stability of the protein. Conventionally, to evaluate the impact of a variant on stability, the variant must be enriched to a reasonable purity, and then its stability characterized by chromatographic or biophysical techniques. However, it is often impractical to purify and characterize each variant in a therapeutic protein. A workflow, based on limited proteolysis followed by MS detection, was established to simultaneously assess the impact of a large number of variants on conformational stability without enrichment. Because a less stable domain is more susceptible to proteolytic degradation, conformational stability of the domain can be reported from the release rate of a proteolytic peptide. A kinetic model is established to quantitatively determine the extent of domain stabilization/destabilization of different variants. The methodology is demonstrated by examining variants known to affect the stability of immunoglobulin domains, such as different N-glycoforms, methionine oxidations, and sequence variants. With this methodology, near 100 variants may be evaluated within 2 days in a single experiment. Insights into the sequence-stability relationship will be obtained by monitoring the large number of low-level sequence variants, facilitating engineering of more stable molecules.

Study on the new dynamics and driving factors of soil pH in the red soil, hilly region of South China.

Soil acidification has always been a substantial eco-environmental problem restricting agricultural development in the red soil region of southern China. It is necessary to determine the dynamic change in soil pH in this area to formulate regional agricultural and environmental management measures. Yujiang County, a typical county with red soil acidification in southern China, was selected as the study area. Based on soil data from 1982, 2007, and 2018, the spatiotemporal variation characteristics and the latest changes in soil pH in the county were analyzed. The results show that the soil pH in Yujiang County decreased from 5.66 to 4.74 and then increased to 4.96 from 1982 to 2018, showing a trend of first decreasing and then increasing. According to the spatial distribution characteristics of soil pH, the low soil pH values in the three periods were mainly distributed in the northern mountainous areas with more forestland and dry land area and some southern hilly areas, while the paddy soil pH values in the middle low hilly areas were relatively higher. The soil pH decreased rapidly from 1982 to 2007, showing a large area of acidification. In 2007, the proportions of acidic (4.5 < pH < 5.5) and strongly acidic (pH < 4.5) soils increased by 67.37% and 10.6%, respectively, compared with that in 1982. However, from 2007 to 2018, the soil pH of the whole county increased, and the acidification trend was alleviated, which is of great significance to the regional red soil ecological environment. Through the analysis of the main factors affecting the change in soil pH, it was found that the sharp decline in soil pH in Yujiang County during 1982-2007 was mainly caused by acid rain and excessive nitrogen application. From 2007 to 2018, no significant reduction in nitrogen fertilizer in this area occurred, and although the increase in soil organic matter contributed to alleviating soil acidification, the analysis showed that the decrease in acid rain was the main reason for the rise in soil pH in Yujiang County. At the same time, notably, there is a large area of soil in the area that is still acidic, and effective control of soil acidification is still an important ecological and environmental issue in this area. In order to further improve the pH value of soil in red soil region, it is suggested that on the basis of continuous improvement of acid rain, in addition to increasing soil organic matter by returning straw to field and other measures, appropriate amount of lime or alkaline biochar can be applied to better improve the soil ecological environment in red soil hilly region.

Complete Extraction of Protein Dynamics Information in Hydrogen/Deuterium Exchange Mass Spectrometry Data.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has been used to study protein conformation and conformational dynamics. A continuous labeling experiment, followed by proteolytic digestion and MS analysis, generates a large amount of data, containing information on protein conformation and conformational dynamics. Lacking appropriate computational methods, information hidden inside the isotope distribution is often omitted and not extracted. In this work, a computational model is described to simulate the determined isotope pattern for each proteolytic peptide at each labeling time. Optimizing the model with experimental data yields conformational protection as well as protein unfolding/folding kinetics. With this method, complete extraction of protein dynamics information in the HDX-MS data is achieved. Information derived from the method is reliable as the model is mostly based on first-principles with very few assumptions. It is demonstrated that the protein dynamics information extracted from one or two labeling time points approaches or exceeds the information derived from an entire deuterium uptake time course by the traditional method. Application of the computational method to an IgG1 antibody under mild denaturing conditions indicates that the unfolding of each immunoglobulin domain can be explained by a simple two-state unfolding process, with different unfolding rate for each domain.

Quantitative structure-chromatographic retention relationship of synthesized peptides (HGRFG, NPNPT) and their derivatives.

Carapax Trionycis extract peptides (HGRFG, NPNPT) are able to protect against CCl4-induced liver fibrosis. Therefore, this study applies to deal with chromatographic lipophilicity determination of synthesized peptides (HGRFG, NPNPT) and their derivatives using reversed-phase high performance liquid chromatography (RP-HPLC) combined with methanol-water mobile phase and two reversed-phase chromatographic columns (COSMOISL 5C18-MS-II and SHIMADZU-C18). The chromatographic lipophilicity of the analyzed compounds was expressed as logkw constant and correlated with lipophilicity descriptors. Quantitative structure-retention relationships (QSRR) analysis was performed to imitate chromatographic lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression (LR) and multiple linear regression (MLR) methods with the help of principal component analysis (PCA) and hierarchical cluster analysis (HCA). The most influential molecular descriptors were lipophilicity descriptors, which are important for molecules ability to pass through biological membranes. All established QSRR models were statistically validated by standards, cross- and external validation parameters. According to these statistical validation parameters, MLR models (R2 > 0.856) were better for chromatographic lipophilicity prediction of peptide compounds. It can be concluded that chromatographic systems with COSMOISL 5C18-MS-II column were better for modeling of logkw than systems with SHIMADZU-C18 column. Modeling was performed in order to obtain lipophilicity profiles of investigated compounds as future drug candidates.

circStrn3 is involved in bone cancer pain regulation in a rat model.

Bone cancer pain (BCP) is a common chronic pain that is caused by a primary or metastatic bone tumor. More detailed molecular mechanisms of BCP are warranted. In this study, we established a BCP rat model. The von Frey hair test, body weight, and hematoxylin and eosin staining were employed. We screened differentially expressed circRNAs (DECs) between the BCP group and sham group. The results revealed that 850 DECs were significantly up-regulated and 644 DECs were significantly down-regulated in the BCP group. Furthermore, we identified 1177 differentially expressed genes (DEGs) significantly up-regulated and 565 DEGs significantly down-regulated in the BCP group. Gene Ontology annotation of all 1742 DEGs revealed that biological regulation of metabolic processes, cellular processes, and binding were the top enriched terms. For Kyoto Encyclopedia of Genes and Genomes analysis, phagosome, HTLV-I infection, proteoglycans in cancer, and herpes simplex infection were significantly enriched in this study. In addition, we identified four selected circRNAs, chr6:72418120|72430205, chr20:7561057|7573740, chr18:69943105|69944476, and chr5:167516581|167558250, by quantitative real time PCR. chr6:72418120|72430205 (circStrn3) was selected for further study based on expression level and the circRNA-miRNA-mRNA network table. Western blot analysis suggested that knockdown of circStrn3 could effectively induce Walker 256 cell apoptosis. In summary, our study provided a more in-depth understanding of the molecular mechanisms of BCP.

Molecular basis for the loss-of-function effects of the Alzheimer's disease-associated R47H variant of the immune receptor TREM2.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.

Efficacy and Safety of Tedizolid Phosphate versus Linezolid in a Randomized Phase 3 Trial in Patients with Acute Bacterial Skin and Skin Structure Infection.

Tedizolid phosphate is approved for the treatment of acute bacterial skin and skin structure infection (ABSSSI) caused by Gram-positive bacteria in the United States, Europe, and other countries. In this multicenter, double-blind, phase 3 study, 598 adult ABSSSI patients in China, Taiwan, the Philippines, and the United States were randomized to receive 200 mg of tedizolid, intravenously (i.v.)/orally (p.o.), once daily for 6 days or 600 mg of linezolid, i.v./p.o. twice daily for 10 days. The primary endpoint was early clinical response rate at 48 to 72 h. Secondary endpoints included programmatic and investigator-assessed outcomes at end-of-therapy (EOT) and posttherapy evaluation (PTE) visits. Safety was also evaluated. In the intent-to-treat (ITT) population, 75.3% of tedizolid-treated patients and 79.9% of linezolid-treated patients were early responders (treatment difference, -4.6%; 95% confidence interval [CI], -11.2, 2.2). After exclusion of patients who never received the study drug (tedizolid, n = 8; linezolid, n = 1; modified ITT), comparable early response rates were observed (tedizolid, 77.4%; linezolid, 80.1%; treatment difference, -2.7%; 95% CI, -9.4, 3.9). Secondary endpoints showed high and similar clinical success rates in the ITT and clinically evaluable (CE) populations at EOT and PTE visits (e.g., CE-PTE for tedizolid, 90.4%; for linezolid, 93.5%). Both drugs were well tolerated, and no death occurred. Eight patients experienced phlebitis with tedizolid while none did with linezolid; hence, drug-related treatment-emergent adverse events were reported in a slightly higher proportion in the tedizolid (20.9%) arm than in the linezolid arm (15.8%). The study demonstrated that tedizolid in a primarily Asian population was an efficacious and well-tolerated treatment option for ABSSSI patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT02066402.).

Reliable LC-MS Multiattribute Method for Biotherapeutics by Run-Time Response Calibration.

A major challenge of a mass-spectrometry-based quantitative multiattribute method (MAM) for biotherapeutics is its high variability between instruments. For reproducible attribute measurements, not only is a similar instrument model required, but the instruments must also be tuned to the same condition. This poses great long-term challenges, considering the rapid development of new instrumentations. In addition, differences in digestion efficiency, peptide recovery, and artificial modifications during sample preparation also contribute to variability between laboratories. To overcome these challenges, new mathematical methods are developed to calculate the attribute abundance in the sample, using the reference standard (RS) material as calibrant. Most quality attributes in the RS remain constant throughout the life of the standard, and therefore, the RS can serve as a calibrant to correct for the difference between instruments or sample preparation procedures. Because RS data are usually collected in a MAM assay, no additional work is required from the analyst. Data from a large number of attributes demonstrated that these methodologies greatly reduced instrument-to-instrument and sample preparation variabilities. With these methodologies, a consistent instrument model and sample preparation procedure is no longer a requirement. As a result, changes in digestion procedure and advances in instrumentations will not significantly affect the assay result.

MS-based conformation analysis of recombinant proteins in design, optimization and development of biopharmaceuticals.

Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics.

Amino Acid Misincorporation Propensities Revealed through Systematic Amino Acid Starvation.

Elevated amino acid misincorporation levels during protein translation can cause disease and adversely impact biopharmaceutical product quality. Our previous work, along with that of others, identified numerous low-level unintended sequence variants. However, because of the limited analytical detection efficiency, we believed that these observations represented only a fraction of biologically relevant outcomes. Because amino acid misincorporation can be exacerbated by amino acid starvation, we believed that a more comprehensive set of sequence variants could be derived through systematic starvation. Our goals for this study were therefore (1) to systematically characterize misincorporation patterns under amino acid starvation and (2) to elucidate the major misincorporation mechanisms and propensities for cultured mammalian cells. To the best of our knowledge, this is the first study to use controlled systematic starvation to maximize the observation of unique sequence variants to provide a more holistic perspective of amino acid misincorporation. Our findings bridge the two prevailing lines of research and propose that both base mismatches during codon recognition (especially G/U and wobble mismatches) and misacylation are common and major amino acid misincorporation mechanisms. This proposal is also supported by the observation of mechanistic additivity between the base mismatch and misacylation mechanisms. In addition, we observed significant overlap in misincorporation mechanisms and propensities among cell lines and organisms. Lastly, we explored factors that can lead to codon-associated misincorporation behavior.

Spatial distribution characteristics of soil organic carbon in subtropical forests of mountain Lushan, China.

The study on the spatial distribution of forest soil organic carbon (SOC) is of great significance for accurate assessment of carbon storage in forest ecosystems. In the present study, by taking eight kinds of forest soils of Mountain Lushan in the subtropical area as the research object, we studied the spatial distribution characteristics of SOC in this mountainous area. The results showed that the SOC content and SOC density (SOCD) of main forest types in the Mountain Lushan were lower than the national and the world average. The soil layer of Lushan forest was thinner, and the SOC and active SOC (ASOC) contents of different forest types and SOCDs are the highest in the surface soil. SOCD of the topsoil accounts for 32.64-54.03% of the total SOCD in the whole soil profile. Surface litter is an important source of SOC, and the different vegetation types are the important reason for the different spatial distribution of SOC in this area. Soil SOC contents in the high-altitude forest (bamboo forest, deciduous broadleaf forest, Pinus taiwanensis forest, evergreen-deciduous forest, and coniferous-broadleaved mixed forest) were higher than those in the low-altitude forest (evergreen broadleaf forest, shrub, and Pinus massoniana forest). However, the difference in SOC content exhibited at the altitude gradient is significantly lower than that in SOC in the soil profile. This indicates that both soil depth and elevation are the important factors that affected SOC distribution. However, the influence of soil depth on spatial distribution of SOC may be more complex than that of altitude. Vegetation types and soil properties are the main reasons for the large differences of reduction rate in the contents of SOC and ASOC.

GhVLN4 is involved in cell elongation via regulation of actin organization.

MAIN CONCLUSION: GhVLN4 exhibited activity of cross-linking actin filaments into bundles. Overexpression of GhVLN4 increased the abundance of thick actin bundles and resulted in longer cell phenotypes. Actin bundle is a dynamic, higher-order cytoskeleton structure that is essential for cell expansion. Villin is one of the major proteins responsible for crosslinking actin filaments into bundles. However, this kind of actin binding protein has rarely been investigated in cotton. In the present work, a cotton villin gene was molecularly cloned from Upland cotton and denominated as GhVLN4. This gene was more highly expressed in fiber-bearing wild-type cotton TM-1 (Texas Marker-1) than in Ligon lintless-1 mutant (Li-1). Biochemical analysis combined with subcellular localization revealed that GhVLN4 is an actin-binding protein performing actin filament bundling activity in vitro. In line with these findings, a greater abundance of thick actin filament bundles were observed in GhVLN4-overexpressing transgenic plants compared with those in wild-type control. Moreover, ectopic expression of GhVLN4 significantly enhanced the cell length-width ratio of Schizosaccharomyces pombe yeast and increased the length of various Arabidopsis cells, including root cells, root hairs and pollen tubes. Taken together, our results demonstrate that GhVLN4 is involved in the generation of actin filament bundles, suggesting that GhVLN4 may play important roles in regulating plant cell morphogenesis and expansion by remodeling actin cytoskeleton.