Phenylpropanoid amides from Solanum rostratum and their phytotoxic activities against Arabidopsis thaliana.

Introduction: Solanum rostratum, an annual malignant weed, has seriously damaged the ecological environment and biodiversity of invasion area. This alien plant gains a competitive advantage by producing some new phytotoxic substances to inhibit the growth of native plants, thus achieving successful invasion. However, the chemical structures, inhibitory functions and action mechanisms of phytotoxic substances of S. rostratum remain unclear. Methods: In this study, to clarify the chemical structures of phytotoxic substances from S. rostratum, we isolated phenylpropanoid amides from the plant. Their structures were identified by comprehensive HR-ESIMS, NMR and ECD data. And the inhibitory functions of isolated phenylpropanoid amides on one model plant (Arabidopsis thaliana) were also investigated. In addition, the action mechanisms of active phenylpropanoid amides were revealed by antioxidant-related enzymes [Catalase (CAT), Peroxidase (POD), Superoxide dismutase (SOD)] activities and corresponding molecular docking analyses. Results and Discussion: Phytochemical research on the whole plant of S. rostratum led to the isolation and identification of four new phenylpropanoid amides (1-4), together with two known analogues (5-6). All the compounds showed phytotoxic effects with varying levels on the seed germination and root elongation of one model plant (Arabidopsis thaliana), especially compound 2 and 4. Likewise, compounds 2 and 4 displayed potent inhibitory effects on antioxidant-related enzyme (POD). In addition, compounds 2 and 4 formed common conventional hydrogen bonds with residues Ala34 and Ser35 in POD revealed by molecular docking analyses. These findings not only helped to reveal the invasion mechanism of S. rostratum from the perspective of "novel weapons hypothesis", but also opened up new ways for the exploitation and utilization of S. rostratum.

Biosimilar or Not: Physicochemical and Biological Characterization of MabThera and Its Two Biosimilar Candidates.

The development of therapeutic biosimilar antibodies has become an important driving force of the modern biopharmaceutical industry. In this study, physiochemical characteristics (amino acid sequence, intact/subunit molecular weight, isoelectric point, post-translation modification, and disulfide linkage pattern), purity (charge variants, high and low molecular weight variants), antigen binding activity, Fc receptor binding affinity and Fc-effector function (CDC and ADCC) were analyzed by using an extensive set of state-of-the-art and orthogonal analytical technologies to provide a comprehensive characterization of the innovative product rituximab and two biosimilar candidates. The similarity study showed that biosimilar candidate 1 (BC1) and the reference product (RP) MabThera had an identical protein amino acid sequences and highly similar primary structures along with similar purity, heterogeneity profiles, antigen binding activity, Fc receptor binding affinity, and Fc-effector functions. Biosimilar candidate 2 (BC2), which had an amino acid replacement at a constant region, a different N-glycosylation profiling, and purity, was not analytically similar to RP. Although BC2 showed improvement such as an increased level of afucose, another IgG1 allotype, and similar biological activities, it was not recommended to be applied as a biosimilar compound in drug registration because the biosimilar manufacturer must first show that its primary structure was identical to that of RP. Our physicochemical characterizations and bioassay comparability study provided a deepened understanding of the structure-function relationship of quality attributes.

Helix Matrix Transformation Combined With Convolutional Neural Network Algorithm for Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Bacterial Identification.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis is a rapid and reliable method for bacterial identification. Classification algorithms, as a critical part of the MALDI-TOF MS analysis approach, have been developed using both traditional algorithms and machine learning algorithms. In this study, a method that combined helix matrix transformation with a convolutional neural network (CNN) algorithm was presented for bacterial identification. A total of 14 bacterial species including 58 strains were selected to create an in-house MALDI-TOF MS spectrum dataset. The 1D array-type MALDI-TOF MS spectrum data were transformed through a helix matrix transformation into matrix-type data, which was fitted during the CNN training. Through the parameter optimization, the threshold for binarization was set as 16 and the final size of a matrix-type data was set as 25 × 25 to obtain a clean dataset with a small size. A CNN model with three convolutional layers was well trained using the dataset to predict bacterial species. The filter sizes for the three convolutional layers were 4, 8, and 16. The kernel size was three and the activation function was the rectified linear unit (ReLU). A back propagation neural network (BPNN) model was created without helix matrix transformation and a convolution layer to demonstrate whether the helix matrix transformation combined with CNN algorithm works better. The areas under the receiver operating characteristic (ROC) curve of the CNN and BPNN models were 0.98 and 0.87, respectively. The accuracies of the CNN and BPNN models were 97.78 ± 0.08 and 86.50 ± 0.01, respectively, with a significant statistical difference (p < 0.001). The results suggested that helix matrix transformation combined with the CNN algorithm enabled the feature extraction of the bacterial MALDI-TOF MS spectrum, which might be a proposed solution to identify bacterial species.

A novel triplex isobaric termini labeling quantitative approach for simultaneously supplying three quantitative sources.

Benefiting from high sensitivity and great ability to measure multiple samples simultaneously, isobaric tandem Mass spectrometry (MS2) quantification has been widely applied for protein biomarker screening. Here, a newly developed isobaric MS2 quantification method named triplex quantification by isobaric termini labeling (Triplex-QITL) was established. This method enables the accurate comparison of various fragment ions (reporter ions, amino acid fragments and N-/C-terminal fragments) based quantification to be operated in a single run. To our knowledge, this is the first time that this kind of comparison is achieved. In Triplex-QITL, proteins were first digested with Lys-C to produce peptides with lysine (K) at the C-termini, then dimethylation reagents and mTRAQ reagents were used to label the N-termini and C-termini of the peptides respectively. N- and C-terminal fragment ion pairs, reporter ions from mTRAQ (113,117,121) and a1 ion pairs were simultaneously generated in MS2 spectra. In simple sample experiment, not much difference in using various fragment ions for quantification was observed. When analyzing SW480 cell lysate, comparing with a1 ions, about two times of reproducible quantification results were achieved by reporter ions and N- and C-terminal ions. Meanwhile the measured quantification results were much closer to the expected results even in large ratios (1:10:10) using N- and C-terminal ions. Finally, Triplex-QITL was successfully applied to profile metastatic differences of three hepatocellular carcinoma (HCC) cell lines. In all, Triplex-QITL shows a promising future in quantitative proteomics.

Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine.

Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

Direct-S: a directed mass spectrometry method for biomarker verification in native serum.

Serum has been the logical choice and most-used bio-specimen for monitoring biomarkers. However, direct analysis of low-abundance biomarkers in serum is still a problem. Here, we have established a directed mass spectrometry (inclusion list driven MS) method, Direct-S, for direct quantification of protein biomarkers in native serum samples without high-abundance protein depletion or pre-fractionation. In Direct-S, an (18)O-labeling technique was used to produce internal standards of the targeted peptides, and only targeted peptides were selected for tandem mass spectrometry (MS/MS) fragmentation to increase sensitivity and efficiency. The (16)O/(18)O ion pairs of target peptides and the elution time/fragmental pattern of the internal standards were used to facilitate the identification of the low-abundance peptides. Using Direct-S, three candidate biomarkers, α1-antitrypsin (A1AT), galectin-3 binding protein (LG3BP) and cathepsin D (CTSD), which represent different abundance levels, were quantified in serum samples of colorectal cancer (CRC) patients and healthy candidates. Direct-S exhibited good linearity of response from 20 fmol to 0.5 nmol (r > 0.9845). Reliable quantification across five orders of magnitude and as low as 71 pg µL(-1) was achieved in serum samples. In conclusion, Direct-S is a low cost, convenient and accurate method for verifying serum biomarkers.

Hyperplex-MRM: a hybrid multiple reaction monitoring method using mTRAQ/iTRAQ labeling for multiplex absolute quantification of human colorectal cancer biomarker.

Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

Recent progress in quantitative glycoproteomics.

Protein glycosylation is acknowledged as one of the major posttranslational modifications that elicit significant effects on protein folding, conformation, distribution, stability, and activity. The changes in glycoprotein abundance, glycosylation degree, and glycan structure are associated with a variety of diseases. Therefore, the quantitative study of glycoproteomics has become a new and popular research topic, and is quickly emerging as an important technique for biomarker discovery. Mass spectrometry-based protein quantification technologies provide a powerful tool for the systematic and quantitative assessment of the quantitative differences in the protein profiles of different samples. Combined with various glycoprotein/glycopeptide enrichment strategies and other glycoprotein analysis methods, these techniques have been further developed for application in quantitative glycoproteomics. A comprehensive quantitative analysis of the glycoproteome in a complex biological sample remains challenging because of the enormous complexity of biological samples, intrinsic characteristics of glycoproteins, and lack of universal quantitative technology. In this review, recently developed technologies in quantitative glycoproteome, especially those focused on two of the most common types of glycosylation (N-linked and O-linked glycoproteome), were summarized. The strengths and weaknesses of the various approaches were also discussed.

Preparation of pH-responsive stationary phase for reversed-phase liquid chromatography and hydrophilic interaction chromatography.

Novel pH-responsive polymer-grafted silica was successfully synthesized through the radical "grafting from" polymerization on azo initiator-immobilized silica. The immobilization of azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4'-azobis(4-cyanovaleric acid chloride). The polymer-grafted silica was prepared by stirring suspension of the azo initiator-immobilized silica in anhydrous dioxane containing acrylic acid (AAc) and butyl acrylate (BA). The resulting polymer-grafted silica was demonstrated to be pH responsive to hydrophobic/hydrophilic property by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). In RPLC mode, the retention of aromatic compounds decreased with the increase in the pH of mobile phase. However, the opposite result was obtained in HILIC mode; the retention of soybean isoflavones was stronger with the mobile phase at higher pH. Finally, the separations of sulfonamides and soybean isoflavones were carried out in RPLC mode and the separation of some nucleotides was achieved in HILIC mode.

Humic acid-bonded silica as a novel sorbent for solid-phase extraction of benzo[a]pyrene in edible oils.

A novel solid-phase extraction (SPE) sorbent, humic acid-bonded silica (HAS), was prepared. Humic acids (HAs) were grafted onto silica matrices via an amide linkage between humyl chloride and the amido terminus of 3-aminopropyltrimethoxysilane (APTS)-silica gel. The resulting material was characterized by Fourier transform infrared spectrometer, elemental analysis, and nitrogen adsorption analysis. This sorbent exhibits an excellent adsorption capacity for some electron-abundant analytes owing to its peculiar structure. In this paper, we choose benzo[a]pyrene (BaP) in oil as a probe to validate the adsorption capacity of the material. Thus a fast, cheap and simple SPE method with humic acid-bonded silica cartridge for edible oil clean-up, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was established. The effects of experimental variables, such as washing and elution solvents, and the amount of sorbents have been studied. The recoveries of BaP in edible oils spiked at 0.2-100 microg kg(-1) were in the range of 78.8-102.7% with relative standard deviations ranging between 1.3 and 9.3%; the limit of detection was -0.06 microg kg(-1).