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With the size of the biopharmaceutical market exponentially increasing, there is an aligned growth in the importance of data-rich analyses, not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships. In monoclonal antibodies, many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known. The importance of their function focuses analytical research efforts on the development of robust, accurate and fast methods to support drug development and quality control. Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however, it is not the only method for quantitative analysis of glycoform heterogeneity. In this study, ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies. While observing good comparability between the quantitative results generated, it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow ac-cording to application and the desired depth of data generated.
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Objective Toinvestigatestatistically significant peptide peaks as biomarkersto diagnose osteoarticular tuberculosis, matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) was applied to identify the characteristic fingerprint among the serum of patients with osteoarticular tuberculosis, rheumatoid arthritis and healthy adults.Methods Clinical Study. Serum samples of untreatedpatients with osteoarticular tuberculosis and rheumatoid arthritis were collected from August 2018 to December 2018, and serum samples of healthy adults from physical examination were collected as control. After analysis with MALDI-TOF MS, the serum peptide fingerprint datawas imported into software, and protein polypeptide peaks with obvious differences were screened to establish diagnostic models.Results Established the diagnostic model of osteoarticular tuberculosis and healthy adults with m/z 2943.9, 5929.6, 7615.4 and 9033.8 as differential protein polypeptides, the diagnostic model of osteoarticular tuberculosis and rheumatoid arthritis with m/z 4195.6, 5847.6, 5929.6 and 7748.6 as differential protein polypeptides. To these two models, the sensitivity were 95.00％ and 97.50％, respectively. The specificity were 85.71％ and 88.46％, respectively. The accuracy rates were 89.58％ and 92.39％, respectively. The AUC value of ROC curves were 0.8859 and 0.8709, respectively. Conclusions By mass spectrometry and software analysis, the serum protein polypeptides with statistical difference were found successfully. The related diagnostic modelsarealso established, which has certain reference value for auxiliary diagnosis of osteoarticular tuberculosis.
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OBJECTIVE: To establish quality control methods for recombinant anti-EBOV mAbs which are comprised by three anti-EBOV glycoprotein mAbs. METHODS: The binding-activity of recombinant humanized anti-EBOV mAbs to EBOV glycoprotein was evaluated by ELISA. Peptide map by LC-MS was used in the identification tests. Purity was analyzed by CE-SDS and SEC-HPLC. iCIEF was performed to measure the PI value and the charge heterogeneity. 2AB was labeled on the released glycan, and was analyzed by NP-HPLC and mass spectrometry. The concentration of polysorbate 20 was tested by HPLC. RESULTS: The EC50s of recombinant anti-EBOV mAbs were (12.53±1.62), (11.10±0.62) and (6.09±0.35) ng·mL-1, respectively. The theoretical sequence coverage rates of three mAbs were all above 95%. The main peak area percentages shown by non-reduced CE-SDS were (94.41±0.05)%, (95.58±0.17)% and (96.11±0.05)%. The peak area percentages of both heavy and light chain shown by reduced CE-SDS were (98.19±0.06)%, (97.97±0.03)% and (98.57±0.03)%. The main peak area percentages shown by SE-HPLC were (99.59±0.01)%, (99.56±0.01)% and (99.74±0.01)%. The isoelectric points of the main peak shown by iCIEF were (8.70±0.01),(8.26±0.01) and (8.85±0.01). The concentrations of polysorbate 20 were (0.34±0.00),(0.35±0.00) and (0.35±0.01) μg·mL-1, respectively. The glycan mapping analysis was relatively sensitive, and the percentage of fucosylated N linked glycan was less than 0.5%. CONCLUSION: Up-to-date quality control methods for recombinant anti-EBOV mAbs are established in this study, which may be used to ensure the safety, effectiveness and quality controllability of the product. The methods can provide technical assists to Ebola outbreak and be a reference of the quality control of other domestic cocktail monoclonal antibody products.
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Starch phosphorylase (EC 2.4.1.1) is the key enzyme which catalyzes the reversible conversion of starch and inorganic phosphate into glucose-1-phosphate (G-1-P) and has been reported in many higher plants. It can be exploited for industrial purposes for tailoring starch and for the synthesis of biopolymers which may be useful in food industry and are also of clinical importance. Starch in the form of insoluble granules accumulates in chloroplasts as the primary product of photosynthesis. Multiple forms of the enzyme have been reported in different plant tissues which have been predicted to have different roles in starch metabolism. Here, various biochemical properties have been reviewed. Various techniques for enzyme immobilization have been discussed. Besides, reports on immobilization of starch phosphorylase from different sources and on different solid matrices have also been reviewed. Peptide mapping reports of various proteins have also been assessed in this review.
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OBJECTIVE: To evaluate the quality status and current standard of cytochrome C injection and to carry out exploratory research on its standard. METHODS: Samples were tested according to the current quality standard in the Pharmacopoeia of the People's Republic of China (2010) Volume II- Exploratory researches including peptide mapping analysis, activity determination by TMB substrate ehromogenic method, and related substances detection by HPLC were carried out. RESULTS: The passing rate of cytochrome C injection was 100%, and that of freeze-dried cytochrome C powder for injections was 50%. CONCLUSION: There existed some problems with both the quality status and current standard of cytochrome C injection. The exploratory researches indicate that it is necessary to improve the statutory standard of cytochrome C.
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Objective To explore the establishment of peptide mapping database of Candida albicans ,laying the foundation for rapid diagnosis of Candida albicans infection .Methods 96 Candida albicans were collected clinically ,and its DNA was extracted . Polymerase chain reaction(PCR) was used to amplify the ITS1-5 .8S-ITS2 gene fragments and restriction endonucleases were a-dopted to identify them .Surface enhanced laser desorption ionization-time of flight-mass spectrometry(SELDI-TOF-MS) instrument was applied to detect the Candida albicans peptide mapping ,and Ciphergen ProteinChip software was used to collect data automati-cally .The established peptide mapping database was verified by confirmed Candida .Results According to restriction fragment length polymorphism analysis ,96 strains were confirmed as Candida albicans .15 peptide peaks were captured by SELDI-TOF-MS chips .Five peptide peaks of them with stable expression were screened out ,and the similarity analysis software was used to estab-lish peptide mapping database of Candida albicans .More than 95% of similarity was found between peptide mapping of Candida albicans and established database ,while less than 50% was found between peptide mapping of other Candida species and database . Conclusion The establishment of peptide mapping database of Candida albicans provides a theoretical basis for the rapid diagnosis of Candida albicans infection .
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OBJECTIVE: To determine pegylation sites of PEG-rhGH by RP-HPLC peptide mapping. METHODS: Firstly, a RP-HPLC method was set up to get high resolution and detailed information of peptides by optimizing the separating and digestion conditions; secondly, MALDI-TOF mass was used to identify each separated peptide; finally, pegylation sites were determined through comparing the k values of changed peptides in the modified and unmodified digestion map. RESULTS: There were five major peptides modified by PEG in the digestion of PEG-rhGH from three pharmaceutical companies. They were T1(N-end), T4(lys38), T13 (lys140), T14(lys145) and T15(lys158), respectively. CONCLUSION: RP-HPLC peptide mapping can be used to determine pegylation sites of complicated and inhomogeneous positional isomers of PEGylated pharmaceuticals. Copyright 2012 by the Chinese Pharmaceutical Association.
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The aim of present work was to investigate the purification of a novel protein (low molecular weight) from Indian cobra Naja naja by Cation exchange chromatography on CM-Sephadex C-25 and followed by Gelfiltration chromatography on Sephadex G-100. Fraction numbers 26, 27, and 28 were obtained from CM Sephadex C-25. From all the fractions, the protein concentration was calculated and it was applied onto the Sephadex G-100 Gelfiltration chromatography. Fraction No-11 obtained from Sephadex G-100 was used for the determination of molecular weight of the short neurotoxins by SDS-PAGE and Matrix- Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). This SDS-PAGE is corresponding to the gel filtration chromatography which resolved a thick band of ~6-7 kDa proteins, and the MALDI-TOF resolved 6668.530, 7447.438 and 19928.929 Da. Further, this fraction was selected for preparative HPLC by using C18 column, in which two major peaks (retention time 30.793 and 32.846) were found. The peak with retention time of 30.793 was preferred for molecular mass determination by MALDI-TOF showed a single sharp peak of 6815.471 Da. It was digested with trypsin enzymatic cleavage, which explored approximately 26 peptides and their masses were renowned. The scores of all these 26 peptides were compared with online mascot analysis and BLAST sequence and it did not match with any other peptides and proteins. Among these 26 peptides, since only two peptides score as 886.648 and 943.690 Da were identical with short neurotoxin -1 from Naja oxiana, and short neurotoxin -3 from Naja mossambica. Moreover the 6815.471 Da protein was used for hemolytic activity and it did not induce RBC lysis. All this observations suggested that the newly purified protein is a short neurotoxin. This essential information will support us to find out the structural information of short neurotoxin for its application in anti-venom development, antitumor and also for analgesic effects.
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Objective To compare the serum peptidome spectrum between nephrotic syndrome patients and normal controls, and to search for their variations. Methods The serum peptide profiling was determined by ClinProt magnetic bead enrichment and matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in 17 mesangial proliferative glomerulonephritis (MsPGN) patients, 12 minimal change nephrotic syndrome (MCNS) patients, 10 membranous nephropathy (MN) patients, 10 focal segmental glomerulosclerosis (FSGS) patients, and 10 healthy volunteers. Results 5 differentially expressed polypeptides were screened out between MsPGN and normal controls (15.28±7.61, P＜0.01). 7 differentially expressed polypeptides were screened out between MCNS and normal controls (2.16±1.59, P＜0.01). 6 differential expressed polypeptides were screened out between MN and normal controls (35.48±13.71, P＜0.01). 5 differential expressed polypeptides were screened out between FSGS and normal controls (18.06±8.07, P＜0.05). The statistical significance was set at P＜0.05. A Genetic Algorithm was used to set up the classification model between patients and normal controls. The model separated MsPGN, MCNS, MN and FSGS group from normal controls with a cross validation of 96.18%, 100%, 98.53% and 94.12%, respectively. The recognition capabilities were 100%. Conclusions The study established the serum peptidome spectrum for nephrotic syndrome by proteomic technology, and provided a new viewpoint to better understand the pathogenesis of nephrotic syndrome.
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Objective: To investigate the physicochemical properties and biological activity of self-prepared fusion protein inducible co-stimulator-Ig. Methods: Acid hydrolysis, edman degradation and peptide mass finger printing were used to determine the amino acid composition, N-terminal 15 amino acid sequences, and peptide mapping. In vivo mixed lymphocyte reaction assay was used for identification of its biological activity. Results: The result of amino acids composition analysis was consistent with the theoretical value of ICOS-Ig. N-terminal 15 amino acid sequences of the product were EINGSANYEMFIFHN, consistent with the theoretical value of ICOS-Ig. Peptide match assay identified six peptides of the product which could match the theoretic maps of ICOS-Ig. ICOS-Ig and CsA noticeably inhibited the proliferation of allo-reactive T cells in vivo. Conclusion: The prepared ICOS-Ig fusion protein has a correct structure and can inhibit the proliferation of allogeneic T cells in vivo, which lays a foundation for quality control of ICOS-Ig fusion protein.
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markers of endometriosis. Its clinical value deserves further investigation.
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Electrophoresis of human plasma yields 4 butyrylcholinesterase (BChE) protein bands, i.e. C1, C2, C3, C4 and in some individuals also an extraband C5+. In addition to that other protein bands called "S" bands are also invariably detected. In order to know whether the C5+ and the "S" bands are related to the BChE protein, we have carried out immunological and peptide mapping studies on these proteins. The immunology approach was done by raising polyclonal antibodies against each protein bands (S1, S2, C4 and C5+) and reacted to the plasma protein bands transferred on nitrocellulose papers. Individual raised antibodies recognized all protein bands studied including the C4, an isozyme of BChE, indicating that the protein bands contain similar epitopes. Several protein bands cathodic to S1 also reacted with the antibodies, suggesting that they are probably fractions of the BChE protein, as well. When individual protein bands were digested with S. aureus V8 toxin and α-chymotrypsin, they revealed a striking similarity in peptide pattern among each other. These studies indicate that the S1, S2 and C5+ protein bands belong to the BChE protein.
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Butyrylcholinesterase , Peptide MappingABSTRACT
Objective To analyze the alterations of serum protein fingerprint in patients with hypertensive disorder complicating pregnancy(HDCP),screen serum biomarker and establish a diagnostic model of HDCP.Methods Surface-enhanced laser desorption lionization-time of flight-mass spectrometry (SELDI-TOF-MS)technology was used to analyze serum including 25 cases of HDCP patients and 30 cases of age-,gravity-and parity-matched healthy term pregnant women on IMAC3-Cu proteinchip before delivery. Biomarker Wizard and Biomarker Pattern software was used to detect protein peaks significantly different between HDCP and controls,and establish a primary diagnostic model of HDCP.This model was further evaluated by blind test using other 16 parts of serum protein fingerprint.Results Ten protein peaks were significantly different at the molecular range of 2000-50 000(P
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The plysical and chemical characteristics of recombinant human GM - CSF (rhGM - CSF) were studied separatly. rhGM - CSF was treated by GdHCl, reduced by DTT, and the disulfide bond was blocked by idoacetamide. The results showed that the samples aren' t homogeneous in UV absorption spectrum and RP-HPLC analysis after treatment by DTT. There was no remarkable differences in the results of the analysis of SDS-PAGE electrophoresis and western blot between treated and untreated rhGM - CSF samples. The effect of GdHCl on GM-CSF was reversible in all above tests; In peptide mapping analysis, the digestion of the samples with blocked disulfide bond by CNBr and protease is more complete than that without any treatment.
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Tubulin has been purified from mung bean seedling by Zn2+-induced polymerization. Both a- and β-subunits of mung bean tubulin are different from those of brain tubulin in electrophoretic mobility, colchicine binding and peptide map. Heterogeneity of mung bean tubulin has also been documented suggesting diversification of tubulin despite its conserved nature in general.
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A simple, reproducible and rapid protocol for the purification of arginine decarboxylase from Cucumis sativus seedlings has been standardised. The purification steps involved ion-exchange chromatography on diethylaminoethyl-cellulose followed by gel filtration on Sephadex G-l 50. The purified enzyme preparation migrated as a single stainable band on Polyacrylamide gels at both basic and acidic pH, but under denaturing and reducing conditions on sodium dodecyl sulphate-polyacrylamide gels resolved into polypeptides of molecular weight 48,000,44,000 and 15,000. However, in the absence of 2-mercaptoethanol on electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, the enzyme moved as single band with a molecular weight of 150,000. Evidence was obtained to indicate that these three polypeptides were probably derived from a single larger molecular weight enzyme. On storage of the purified protein, the 48,000 species was preferentially degraded to smaller polypeptides. The preliminary data suggested that the 48,000 and 44,000 species shared many common tryptic peptides as revealed by finger printing of the [125I ]-labelled protein. The purified enzyme was a glycoprotein and had a Km of 0·5 mM for arginine. Its activity was stimulated by dithiothrietol and pyridoxal phosphate. EDTA did not inhibit the enzyme activity. Mn2+ at 1 mM stimulated arginine decarboxylase activity but was inhibitory at higher concentration.