Matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry ( MALDI-TOF/ TOF MS) and electrospray ionization-quadrupole-time of flight mass spectrometry ( ESI-Q-TOF MS) were used to confirm the structure of cyclic lipopeptide daptomycin fastly. First, the relative molecular weight 1916.7107 of daptomycin was measured by ESI with error 0.0007. The sample' s doubly charged peak m/z 809.848 was selected as precursor ion for ESI-MS/MS analysis, and the exocyclic amino acid sequence C(9)H(19)C0-Trp-Asn-Asp was successfully matched. Second, the experimental conditions of cleaving daptomycin by lithium hydroxide ( LiOH) were optimized and the ring-opened process was monitored by MALDI-TOF/TOF MS. After obtaining ring-opened product with purity of above 95%, the MS/MS measurements by MALDI and ESI were carried out. The Wand y of ring-opened product were completely matched, which confirmed the amino acid sequence of daptomycin. Finally, ESI-MS/MS conditions of ring-opened product were further optimized to obtain more low mass fragment ions for analyzing the structure of fatty acid chain and the cleavage pattern of fat chain in mass spectrometry was proposed. The method was fast, convenient, accurate and reliable for identifying cyclic lipopeptide compounds.

A turbidimetric method which could verge on a homogeneous reaction system was obtained.The method was applied in study of recombinant human lysozyme activity,and the related operation procedures and quality standards were established.In this research,microscopic counting method was used for the quality control of substrate bacteria liquid.The mixing manner of enzyme and bacteria liquid was improved,so as the amount of enzyme used for detection.Double line methodwas adopted to optimize the reaction time interval for activity detection.Also,the content of recombinant human lysozyme was detected accurately.By means of microscope count method,the bacteria number in reaction substrate was kept at about 1.1 × 108/mL,and the concentration of the reaction substrate was 0.3 mg/mL.To approach the homogeneous system,the reaction liquid of lysozyme and bacteria was mixed by sucking-expelling method in the sample pool.The optimum amount of lysozyme was 3 μg,the optimum reaction time was confirmed as 0-60 s.According to the operation procedure of enzymatic activity detection,the standard for activity of recombinant human lyso-zyme was confirmed as 45 000-65 000 U/mg,the RSD of intra-day and inter-day were both less than 5%.Lowry method was chosen for the content determination of recombinant human lysozyme,and the protein content was(1.0 ± 0.1) mg/mL.Stability of the optimized method was improved obviously,also,the operation was convenient and the detection result was reliable.Furthermore,the optimized method was verified and accepted by three research institutes and enterprises.The quality standards and operational procedures were established with important practical reference value.

Objective To establish an enzyme-linked immunosorbent assay(ELISA)for polypeptide5(Pep5)in human serum and apply it to the serological diagnosis of the progression of liver injury.Methods Microplate was precoated with sample diluents,blocked with 5% milk,and samples were detected with anti Pep5 McAb labled with HRP.And 690clinical samples were detected by method metioned above.Results Sensitivity was 1.25ng/mL.Linear measurement range was 0-24ng/mL.Recovery rate was 99.09%-107.08%.Intra-and inter-assay coefficinet of variation were 7.32%and 6.45%respectively,or less.The cross-reaction rate with acetylcholinesterase,laminin,hyaluronic and procollagen were all less than 1.00%.Clinical detecting showed that levels of Pep5increased with the exacerbation of liver injury(P<0.01).Compared with acetylcholinesterase kit,the relative coefficient(r)was 0.68.Conclusion Pep5could be used for the quantitative serological tests for the progression of liver injury,which might be reliable,simple and convenient,and could provide scientific evidences for the further study of Pep5function and development of related diagnostic kits.