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Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy. Since the expiration of the patent of the innovator epoetin alfa, numerous rhEPO products have emerged in global markets. As described here, multiple complementary analytical approaches are utilized for the extensive characterization of rhEPO molecules, and more importantly for the structural comparison of the rhEPO analogues on the Chinese market. The focus of this study is placed on the overall glycosylation profiling, O-glycan profiling, and N-glycan mapping by UPLC-MS with an aim to develop an effective analytical methodology to monitor the product quality attributes of rhEPO analogues. Two rhEPO analogues manufactured in China were analyzed to demonstrate the principle of the developed methods. Each rhEPO product showed a characteristic glycoform profile with respect to the distribution of sialic acids across multi-antennary structures, the occurrence of O-glycosylation, O-acetylation on sialic acids, and the extension of N-glycan antennae with N-acetyllactosamine units. The study demonstrates that UPLC-MS is an effective analytical tool to characterize and monitor the glycosylation profiles among rhEPO analogues in order to detect and account for the divergence between rhEPO products, as well as the presence of unusual or unexpected glycans. Image 1 • An extensive UPLC-MS characterization of rhEPO molecules for the structural comparison of the rhEPO analogues on Chinese market. • The study focuses on overall glycosylation profiling, O-glycan profiling, peptide mapping, and N-glycan mapping of rhEPO analogues. • An analytical methodology to monitor and assess the product quality attributes of rhEPO analogues is proposed. [ABSTRACT FROM AUTHOR]

Background: Chinese mutton ham is a dry-cured meat product with a long ripening time. The aim of this study was to identify and characterize antioxidant peptides from Chinese mutton ham., Results: Mutton ham peptides (MHPs) were purified by gel filtration, anion exchange and reversed-phase high-performance liquid chromatography steps. The 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging capacity was used to guide the purification of MHPs. Three antioxidant peptides were identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) as Met-Trp-Thr-Asp (MWTD), Ala-Pro-Tyr-Met-Met (APYMM) and Phe-Trp-Ile-Ile-Glu (FWIIE), with molecular weights 551.61, 611.76, and 706.84 Da, respectively. Among them, APYMM exhibited the highest ABTS radical scavenging activity. The three peptides had the ability to inhibit lipid oxidation and Fenton's reagent-induced protein oxidation and DNA damage. After simulated gastrointestinal digestion, FWIIE and APYMM showed increased antioxidant activity, while MWTD showed decreased activity., Conclusion: Three novel peptides isolated from Chinese mutton ham had strong biological activity. Chinese mutton ham is potentially a functional food and an excellent source of natural antioxidants. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

We studied the taxonomic position of six phenetically related strains of the genus Acinetobacter, which were recovered from hospital sewage in China and showed different patterns of resistance to clinically important antibiotics. Whole-genome sequencing of these strains and genus-wide phylogeny reconstruction based on a set of 107 Acinetobacter core genes indicated that they formed a separate and internally cohesive clade within the genus. The average nucleotide identity based on BLAST and digital DNA-DNA hybridization values between the six new genomes were 97.25-98.67% and 79.2-89.3%, respectively, whereas those between them and the genomes of the known species were ≤78.57% and ≤28.5%, respectively. The distinctness of the strains at the species level was also supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by MALDI-TOF MS. Moreover, the strains displayed a catabolically unique profile and could be differentiated from the phylogenetically closest species at least by their inability to grow on d,l-lactate. A total of 18 different genes were found in the six genome sequences which encode resistance to seven classes of antimicrobial agents, including clinically important carbapenems, oxyimino-cephalosporins, or aminoglycosides. These genes occurred in five different combinations, with three to 10 different genes per strain. We conclude that the six strains represent a novel Acinetobacter species, for which we propose the name Acinetobacter cumulans sp. nov. to reflect its ability to acquire and cumulate diverse resistance determinants. The type strain is WCHAc060092 T (ANC 5797 T =CCTCC AB 2018119 T =GDMCC 1.1380 T =KCTC 62576 T )., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Objective: To investigate the differential protein expression before and after menopause in women with nonalcoholic fatty liver disease (NAFLD) and to explore novel markers for menopausal NAFLD., Methods: Eight serum samples collected from pre- or post-menopausal women with NAFLD were analysed by iTRAQ 2D-LC-MS/MS. Two protein candidates were selected and verified by enzyme-linked immunosorbent assay (ELISA) in serum samples collected from a one hundred and fifty-three female population subsequently, including 51 in post-menopausal status with NAFLD, 41 in pre-menopausal with NAFLD, 19 healthy individuals in post-menopausal status and 42 healthy pre-menopausal women., Results: A total of one hundred and sixty-seven proteins exhibiting significant changes were characterized, among which sixty-five were up-regulated and one hundred and two were down-regulated. Of those altered proteins, the expression of serum retinol binding protein 4 (RBP4) and galectin-3 binding protein (LGALS3BP) was obviously increased in the post-menopausal patient group compared to the other. ELISA validations for the two proteins were consistent with the proteomic profiling., Conclusions: Serum RBP4 and LGAL3BP were up-regulated after menopause under NAFLD conditions, which suggested the two proteins may be potential markers as NAFLD in postmenopausal population., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Cottonseed meal, an important source of feed raw materials, has limited use in the feed industry because of the presence of the highly toxic gossypol. The aim of the current work was to isolate the gossypol-degrading fungus from a soil microcosm and investigate the proteins involved in gossypol degradation., Results: A fungal strain, AN-1, that uses gossypol as its sole carbon source was isolated and identified as Aspergillus niger. A large number of intracellular proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no significant difference was observed between the glucose-containing and gossypol-containing mycelium extracts. Two-dimensional gel electrophoresis results showed that the protein spots were concentrated in the 25.0-66.2 kDa range and distributed in different pI gradients. PDQuest software showed that 51 protein spots in the gels were differentially expressed. Of these, 20 differential protein spots, including six special spots expressed in gossypol, were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry., Conclusion: The fungus AN-1 biodegraded gossypol and the proteomic analysis results indicate that some proteins were involved in the gossypol biodegradation during fungus survival, using gossypol as its sole carbon source., (Copyright © 2011 Society of Chemical Industry.)

Proteomic fingerprint technology combining magnetic beads with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to profile and compare the serum proteins from 45 patients with gallbladder cancer and 50 healthy blood donors. The proteomic patterns were identified; the tree model of biomarkers was constructed and evaluated using the Biomarker Patterns Software. The model tree was constructed based on the 3 biomarkers (5913 Da, 6181 Da and 13,752 Da), which generated excellent separation between the gallbladder cancer and control groups. The sensitivity was 86.7% and the specificity was 93.3%. The blind test data showed a sensitivity of 80% and a specificity of 90%. Taken together, our studies suggested that biomarkers for gallbladder cancer could be discovered in serum by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry combined with the use of magnetic beads. The pattern of combined markers would provide a powerful and reliable diagnostic method for gallbladder cancer with high sensitivity and specificity.

Early detection and diagnosis of colorectal cancer (CRC) are closely related to a better therapeutic outcome, and the five-year survival rate of early CRC is over 90 percent. Though endoscopic minimally invasive treatment has become a quick and effective therapy for early CRC, endoscopic biopsies are usually not deep enough to obtain tissues from the submucosal layer and it is difficult to determine whether early CRC has infiltrated into the submucosa. Therefore, in the present study, we constructed tumor models of early submucosal non-invasive CRC (SNICRC) and submucosal invasive CRC (SICRC) in Fischer-344 rats induced by N-methyl-N-nitrosourea (MNU). The differentially-expressed proteins were analyzed and identified in SNICRC, SICRC and normal control (NC) tissues using highly sensitive two dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). Proteomic data revealed 132 protein spots between SNICRC and SICRC, 162 protein spots between SICRC and NC and 154 protein spots between SNICRC and NC which were found differentially expressed. These differential spots were picked, in-gel digested and peptide mass fingerprints were obtained by MALDITOF-MS/MS. Finally, five differentially-expressed proteins in SNICRC, SICRC and NC were identified, and increases in Transgelin, peptidylprolyl isomerase A (PPIA) and tropomyosin alpha isoform d were observed, while decreases in carbonic anhydrase 2 (CAII) and an unnamed protein were detected in SICRC compared with SNICRC and NC. Furthermore, Fluorescence-based quantitative polymerase chain reaction (FQ-PCR), Western blotting and immunohistochemistry assays also revealed significant upregulation of Transgelin expression and down-regulation of CAII expression in SICRC tissues. In conclusion, 2D-DIGE is confirmed to be an efficient strategy that enables us to identify differentially expressed proteins between early SNICRC and SICRC. The potential biomarkers such as Transgelin and CAII may be used for the detection of early SICRC.

A fingerprint analysis method was developed for Ginkgo biloba leaves and was successfully used for quality evaluation of related health foods by HPLC with electrospray ionization MS. Fifteen samples of G. biloba leaves, which were collected from 15 different locations in China, were analyzed and identified in this study. By both peak analysis and similarity analysis of the fingerprint chromatograms, variation of constituents was easily observed in the leaves from different sources. By comparison with batches of authentic leaves, the authenticity, and quality consistency of related health foods in different matrixes were effectively estimated. It is important to mention that studying a wide range of authentic leaves from various habitats made the quality evaluation of commercial products more convincing and reasonable. The fingerprint-based strategy of the developed method should provide improved QC of G. biloba leaves and products.

Objective: To explore the specific and sensitive biomarkers for gastric cancer detection, a surface-enhanced laser desorption and ionization protein chip mass spectrometry (SELDI-TOF-MS) was used to generate protein profiles of serum in gastric cancer at a high-risk area., Methods: A total of 36 gastric cancer cases and 46 subjects with superficial gastritis were selected from Linqu county, Shandong province, a high-risk area of gastric cancer. Serum samples were collected and Q10 protein chips were used to detect the serum proteomic patterns, and the sensitivity and specificity were assessed., Results: For the comparison of the gastric cancer group (26 out of 36 gastric cancer cases) versus superficial gastritis group (37 out of 46 subjects), the 6 most discriminating peaks (m/z 8587, 6945, 8243, 3899, 7035, and 9943) were identified by the ProteinChip Data Analysis System (ZUCIPDAS). The sensitivity and specificity of this pattern were 88.5% and 97.3%, respectively. A total of 19 subjects (10 gastric cancer cases and 9 superficial gastritis subjects) was selected to test the accuracy of this pattern by using blind method, and the sensitivity and specificity were 80.0% and 88.9% ,respectively., Conclusion: Our findings suggest that SELDI profiling of serum might be a potential for gastric cancer detection and screening in high-risk population.

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 (China isolate), named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus (China isolate). Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DH10Bac. The recombinant bacmid baculoviruses (rBacmid-EGFP-NS3) isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BmN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.

A novel Mesorhizobium group associated with Albizia kalkora [Wang et al. (2006), Syst Appl Microbiol 29, 502-517] was further characterized. The seven strains in this group showed similar protein patterns and were different from defined Mesorhizobium species in SDS-PAGE of whole-cell proteins. The representative strain CCBAU 61158(T) formed a novel Mesorhizobium lineage in phylogenetic analyses of 16S rRNA, atpD, glnII and nifH genes. However, its nodC gene sequence was more similar to that of Rhizobium gallicum R602sp(T) than to those of Mesorhizobium species. DNA-DNA relatedness between CCBAU 61158(T) and reference strains of defined Mesorhizobium species was lower than 34.1 %. These results indicated that this Mesorhizobium group was a unique genomic species. The subtropical distribution, host origin, PCR-RFLP patterns of 16S rRNA genes, fatty acid profile and a series of phenotypic characteristics could be used as distinctive features of this group. This group is therefore proposed as a novel species, Mesorhizobium albiziae sp. nov., with CCBAU 61158(T) (=LMG 23507(T)=USDA 4964(T)) as the type strain. Strain CCBAU 61158(T) could form effective nodules on Albizia julibrissin, Glycine max, Leucaena leucocephala and Phaseolus vulgaris.

Objective: To analyze the alterations of serum proteomic pattern in esophageal squamous cell carcinoma (ESCC) by SELDI-TOF-MS, to establish a diagnostic model of ESCC screening in high incidence area and investigate its clinical value., Methods: SELDI-TOF-MS and CM10 proteinChip were used to detect the serum proteomic patterns of 36 cases of ESCC and 38 healthy control subjects in high incidence area. The data were analyzed and a diagnostic model was established by using support vector machine (SVM). The diagnostic model was evaluated by leave-one-out cross validation., Results: At the molecular weight range of 2000 to 20,000, 31 protein peaks were significantly different between ESCC and controls (P < 0.01). A diagnostic model consisting of 4 protein peaks could do the best in diagnosis of ESCC and controls. The accuracy was 85.1%, sensitivity was 86.1%, specificity was 84.2%, and positive value was 83.8%., Conclusion: The diagnostic model formed by 4 protein peaks, established in this study, can well distinguish ESCC from healthy subjects. It provides a new approach for ESCC screening in high incidence area.

Three new variants of acidic proline-rich proteins (At, Au, Aw) were found in human parotid saliva by isoelectric focusing and basic gel electrophoresis. Electrophoretic comparison of the purified proteins and their tryptic peptides suggested minor charge and size differences from other acidic PRPs. Genetic and biochemical studies indicate that the At and Aw proteins are allelic products of the PRH1 locus. Gene frequencies of the At productive allele (PRH1(6)) in Japanese, Chinese, and Malays were 0.008, 0.012, and 0.004, respectively. The Au protein was also found in Japanese (2 in 746 samples), Chinese (1 in 215 samples), and Malays (1 in 220 samples), however, the Aw protein was found only in one Japanese (n = 746). These three proteins were not found in 106 Indian subjects.