The application effect of isoelectric points(pI) of peptide and protein drugs containing different molecular weights and complex charge heterogeneity was evaluated by capillary isoelectric focusing electrophoresis-whole column imaging detection(cIEF-WCID).The pI of standard polypeptide was mensured repeatedly to verify that cIEF-WCID had a high accuracy and a good repeatability (RSD ＜ 0.50％).Four serum protein isomers had achieved baseline separation.Gan Lai insulin was detected only a single characteristic peaks(pI 5.95 ±0.01) when repeating the test.The proportion differences of the characteristic peak'PI were siguificant,when comparing the recombinant human growth hormone bulk and final,and the final appeared new characteristic peaks.Comparing the imported and domestic Avastin,manufacturer 1 and 3 were found to be consistent with the innovator drug,and the migration behavior of main component in manufacturer 1 was highly consistent with the innovator drug,however,the charge heterogeneity of manufacturer 2 was influenced because of lose K,N-term pyro-Glu.The optimized pI detecting conditions of the recombinant human follicle stimulating hormone were obtained by studying co-solvent concentration,electrolyte and focusing time,namely Co-solvent:2 mol/L urea,ampholytes at pH 2.5-5.0 and pH 3.0-10.0 mixing by 1 ∶ 1,and focus voltage:1 000 V(1 min) to 1 800 V(4 min) and to 2 200 V(1 min).cIEF-WCID was fast and accurate,and it owned excellent resolution and reproducibility,especially,it could trace the focusing process of the analytes.Therefore,it was fit for analysing complex charge heterogeneity of protein drugs.

The relative contents of four kinds of glycan forms in the inovator and biosimilar products of recombinant human thromboplastin (rhTPO) were accurately determined using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) with quasi-internal calibration,the consistencies between products and among batches were evaluated.The N-glycan,sialic acid and O-glycan of rhTPO were removed off with different endoglycosidase,respectively,bovine serum albumin (BSA)used as the standard was pointed target separate with the sample.The molecular weights of the intact glycoproteins and the different forms of deglycosylated proteins were determined by MALDI-TOF MS with quasi-internal calibration.The glycan contents in samples were calculated by the molecular weight differences among different deglycosylated samples.Finally,the consistency of the inovator and biosimilar products were evaluated.The multi-charge peaks of BSA were used for the quasi-internal calibration of MALDI-TOF MS.And their calibration relative standard deviations(RSDs) were all no more than 0.075％,and the RSDs among different calibrations were among 0.001％-0.004％.The method deviations away from the sequence molecular weight were less than 0.01％.For the biosimilar drug,the relative contents of N-glycan,sialic acid,O-glycan,and glycation were 24.6％,2.9％,9.0％ and 5.1％,respectively,and the batch-to-batch consistency was quite good.The total glycan contents were consistent with those reported in the literature.For the innovator drug,the relative contents of N-glycan,sialic acid,O-glycan and glycation were 25.6％,2.9％,7.9％ and 3.5％,respectively.The total glycan content was lower than that reported in the literature.The relative contents of glycosylation between the innoventor products and the biosimilar products were almost the same but the relative contents of glycation showed some differences.Glycation of rhTPO has not been reported up-to-now,the differences of the relative contents of glycation might be the important factors for the different rhTPO manufacturers,the glycation differences were mainly caused by the differences of fermentation process.

Objective Studies on the compliance of recombinant human serum albumin/erythropoietin fusion protein (rHSA/EPO) manufacture process and on the physicochemical properties and propose as basis and technical indicators of quality as a long acting, bio-better innovative protein drug. Methods rHSA/EPO with long-acting and bio-better function was stably expressed in CHO cell line which constructed by genetically engineering technology. A manufacture process was established in a batch scale, suspension and growth in a serum-free medium. Then it was analyzed of the preparation process, detailed physicochemical properties and characteristics of technical quality indicators as a novel drug. Results The purity of rHSA/EPO protein reached more than 97%. Products of rHSA/EPO digested by 3 different binding sites of protease separately and was analyzed with peptide mass fingerprint by MALDI-TOF/TOF PMF and Nano LC-MS/MS with the overall amino acid sequence coverage of 100%. N-terminal amino acid sequence of the first 15 amino acids is identical to the theoretical sequence. The glycosylation sites of rHSA/EPO was positioned with its molecular weight (MW) in mass spectrometry as 93 kD, but the MW of the fusion protein without sugars was 84205 D similar with the theoretical MW of 84849.52 D. The fusion protein contained 1 free sulfhydryl groups, which indicated that there existed only 1 free cysteine in the structure. HSA and EPO on their two space conformation unchanged after a round of two color spectrum analysis. A plurality of charge isomers were in the range of pI 5.1 - 5.8 by a capillary electrophoresis with isoelectric focusing obtained (pI) as about pI 4.9. A typical protein spectrum was obtained by purple spectra. Residues of bacterial endotoxin, host protein, exogenous DNA were in line with the requirements of the regulations as drug raw materials. Immunological identification was positive with rhEPO which also expressed in CHO cells. Biological activity were determination with UT7 cellsin vitro and with micein vivo, but the activity were different. Thein vivo and in vitro bioassay method was used for different experimental purposes, and each also had a certain correlation coefficient. Conclusion The more comprehensive pharmacy data are obtained through studying on the physicochemical properties of the fusion protein. The results can be used as the guidance of the manufacture "Recombinant human serum albumin/erythropoietin fusion protein for injection" (CHO cells) on raw material medicine and preparation and show that the newly created molecular structure of rHSA/EPO (without a linker in between) is comply with the requirements as a bio-better novel protein drug.

Objective To set up a method of stools protein extraction,analysis and identification in order to get the new nonin-vasive indicators of human digestive diseases.Methods The stools proteins,collected from healthy persons,the patients with atrophic gastritis,those who suffed from gastric carcinoma and postoperative patients with gastric carcinoma respectively, were extracted in three different ways including saline,Tris-HCl buffer and Urea buffer,the best way was selected by using SDS-PAGE,then a preliminary analysis of stools proteins was performed by 1D LC-MS/MS.Results The methods of saline and Tris-HCl buffer could get more stools proteins than the method of urea.The proteins in stools from the healthy persons, the patients with atrophic gastritis,the patients with gastric carcinoma and postoperative patients with gastric carcinoma were all abundant and more than one hundred.There was a significant difference in stools protein maps among the various populations.Alpha1-antitrypsin,a number of immunoglobulin and keratin were identified in the stools from patients with gastric carcinoma but not postoperative patients with gastric carcinoma and the healthy persons.Conclusions In this re-search,there was a significant difference in stools protein maps among the healthy persons,the patients with atrophic gastri-tis,the patients with gastric carcinoma and postoperative patients with gastric carcinoma,not only the composition of stools proteins,but also the abundance of same proteins.Therefore,using proteomics technologies to screening of the noninvasive indicators in human stools is viable.The study recommended that the noninvasive indicators in human stools should be iden-tified with quantitative differences analysis combination of quality of mass spectrometer method in the future research.

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.

In order to develop an efficient protein extraction method suitable for tobacco proteome analysis,four protocols for total protein extraction in tobacco root,lysis-buffer method,TCA/acetone precipitation method,Trizol extraction method and phenol method were assessed with tobacco cultivar K326 root at growth stage as experiment materials.The protein samples were separated by SDS-PAGE and two-dimensional electrophoresis(2-DE).It was found that the phenol method was the best one resulting in the most protein bands and the best resolution in SDS-PAGE.About 548 clear protein spots were detected by 2-DE and silver staining with isoelectric points at pH 5～8,and relative molecular weight of protein ranged from 25.0 kD to 70.0 kD.

Objective The regulation of brain Mn depends largely on the blood-brain barrier and blood-cerebrospinal fluid barrier(BCB).The latter is constituted by choroid plexus(CP) epithelial cells,which is specialized for cerebrospinal fluid(CSF) production,has been considered as a primary target in Mn-induced neurotoxicity.This study aims to explore the differential proteome in CSF after sub-acute and sub-chronic Mn exposure in SD rats.Methods(1) Establishment of Mn-animal Model: Rats(1.5 month) were divided into 6 groups;each received daily ip injections of either MnCl2(6 mg Mn/kg BW) or saline(as controls) for 30days(sub-acute),90 days(sub-chronic),or 90 days followed by additional 30-day convalescence.The CSF were collected and analyzed.(2) Evaluation of Mn-animal model(data not shown here,which will be published in another paper).(3) Mn-related proteome in CSF was analyzed by a novel proteomic technique of label-free nanoHPLC-Q-TOF-MS/MS.Results A total of 123 Mn-related differential proteins in CSF were identified,of which 55 were up-regulated,68 were down-regulated.These identified differential proteins could be divided into three categories according to their characteristic change fold(Mn vs.Control):(1) 4 proteins' expression increased by change fold > 1.5,(2) 2 porteins' expression decreased by change fold > 1.5,(3) 66 proteins were only detected in the control group,(4) 51 proteins were only detected in the Mn groups.Based on the information of GO categories,these differentially expressed proteins were mainly from the nuclei,involving in the function of binding;however,more than half of the proteins have no characterized biological function.Conclusion Considering of majority of proteins in CSF being produced and excreted by CP,these differential proteins may be valuable for exploring the novel potential biomarkers to diagnose and monitor the progression of Mn-induced neurodegenerative disease clinically;meanwhile,the results shed light on the future molecular mechanism study of Mn on choroids plexus epithelial cells.

Serum peptides are the rich sources for cancer-specific diagnostic information and because of their complexity, low abundance, degradation, a lot of new peptidomic technologies were developed in recent years. A method based on peptide antibodies and immuno-mass spectrometry for detection of the serum peptide biomarkers was developed for detecting peptide biomarker candidates in the clinic tumor sera. The linear range of the method was 1-40 pmol with a detection limit of 0.5 pmol. The relative standard deviation was 9.5% for 5 pmol standard of synthetic peptide 5(n = 3). Furthermore, this method was successfully applied to determine the serum peptide biaomarker of 35.2 pmol/mL. The method of immuno-mass spectrometry combining peptide antibodies was paticular suitable for the detection of low concentration biomarkers in clinical samples, and it will be significant for earlier diagnosis of cancers.

蛋白质组学是后基因组时代生命科学研究的热点和前沿领域.军事医学科学院是国内最早开展蛋白质组研究的单位之一,其蛋白质组学研究的发展不仅对中国蛋白质组研究起到重要的引领作用,也对国际蛋白质组学的发展做出了重要贡献.本文将重点介绍军事医学科学院的科学家在国际人类肝脏蛋白质组计划以及疾病蛋白质组、病原微生物蛋白质组等领域的研究成果.

Objective This study was designed to investigate if sub-chronic manganese(Mn) exposure led to changes of protein expression and structural damage in the choroid plexus(CP),a brain tissue critical to material transport by the blood-CSF barrier(BCB).Mn-toxicity-related differential expressed proteins in the CP were further analyzed according to Gene Ontology(GO) annotation by cellular distribution,molecular function and biological process.Method Male rats(1.5 months old) were received daily ip injection of either MnCl2(6 mg Mn /kg) or saline(as controls) for 30,90 or 90 days allowed for additional 30-day convalescence.The CP tissues in lateral ventricles were collected at each time point.Result Light microscopy and transmission electron microscopy revealed that Mn exposure resulted in flattened and shrunken cell layer,cytoplasmic vacuolation,nuclei and chromosome condensation,mitochondrion destruction,microvilli shortening,and partial disconnection in intracellular junctions between two adjacent epithelial cells.The structural alteration was mild after 30-day exposure and moderate in 90-day animals.Noticeably,the intracellular damage after 30-day convalescence was even worse than that at 90 days,suggesting a long lasting pathological damage even after Mn exposure was ceased.A total of 32 differential proteins were indentified by ZD-PAGE combined with Nano-LC-MS /MS,of which 27 were up-regulated,5 were down-regulated.Based on the information of GO categories,these differentially expressed proteins mainly locate in the mitochondria,membrane surface and cytoplasm,involving in the function of binding,catalytic activity and transportation and playing a critical role in the biological processes of metabolism and transport.Further verification and analysis are in progress.Conclusion Taken together,these data suggest that Mn not only damages the structural integrity of CP,but also alters the expressions of proteins critical to BCB function.

With the improvement of the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and the expansion of its application fields, the imaging mass spectrometry (IMS) based on MALDI-TOF has become a powerful tool for studying tumor markers, drug metabolism, lipid distribution and so on. The latest sample pre-treatment methods and auto matrix-coating technologies have greatly increased the sensitivity and resolution of IMS. Direct identification of proteins and peptides on tissues by in-situ enzyme digestion is an important focus and key point of IMS. Distribution of drug metabolites and lipid, and IMS for plant tissues are the new fields of applications. This paper reviews systematically the advances of the research and applications of IMS.

Label free LC-MS/MS method was used in seeking of unstable angina blood stasis syndrome(UABSS) differentially expressed plasma proteins. A polyclonal antibody affinity column, nanoAcquity UPLC and Synapt HDMS were used on plasma of two classes of samples(12 UABSS patients and 12 health volunteers). The results show that the method has a good reproducibility. The ionic strength coefficient of variability was less than 5%, retention time coefficient of variability was less than 3%. 3843 proteins were detected, among which 24 kinds of proteins differentially expressed large than 1.5 fold, include 11 down-regulated and 13 upregulated (include 3 kinds of proteins only found in UABSS patients). The dynamic range was about 104. ACTA1; ITIH3 and LBP were only found in the plasma of UABSS patients, Haptoglobin, SAA, CP, C6, MYH11, APOH and ANXA6 were significantly highly expressed in the plasma of UABSS patients, while HBB, HBA, HBE, HBD, HBG, HRG, IGHG, GSN and TF were lowly expressed in the plasma of UABSS patients. These identified increased expressed proteins could be divided into four categories according to their functions: (1) acute phase reactive protein; (2) complement protein; (3) cytoskeletal protein; (4) blood coagulation protein. The decreased expressed proteins could be divided into five categories: (1) apolipoprotein; (2) transport protein; (3) anticoagulated blood protein; (4) immunoglobulin; (5) cytoskeletal modulin protein. In conclusion, UABSS may correlated with inflammatory reaction, lipid metabolic disorder, myocardial damage, blood coagulation factor abnormal, oxygen transport obstacle, and these differentially expressed proteins could provide clues for the study and discovery of new protein targets for antianginal drugs. The label free proteomics is an efficient method for the discovery of differentially expressed proteins of complicated sample.

脉络丛（choroid plexus,CP）位于血液与脑脊液（cerebrospinal fluid,CSF）之间,不仅是CSF的重要来源,而且是构成血液-脑脊液屏障（blood-cerebrospinal fluid barrier,BCB）的组织基础.CP参与脑组织中一些血源性多肽的输送以及自身多肽合成的生理过程,在维持脑微环境动态平衡和调节中枢神经系统的正常功能方面起到非常重要的作用.本研究分别运用MALDI-TOF/TOF和LC-MS/MS液质联用系统分析了成年SD大鼠血液-脑脊液屏障（即脉络丛组织）中的多肽组.共鉴定到163个多肽（P〈0.001）,这些多肽为69种蛋白质的降解肽段,其中ATP合酶（ATP synthase）,细胞色素c（cytochrome c）,血红蛋白（hemoglobin）,NADH-辅酶Q氧化还原酶（NADH-ubiquinone oxidoreductase）,β珠蛋白（beta-globin）这5种蛋白质的肽段数占总肽段数的50%以上,并且部分多肽序列相似度高,类似其前体蛋白的逐步降解片段,而这些前体蛋白质的分子量多数在10kD至20kD之间.上述研究结果为SD大鼠脉络丛组织的生理功能研究及组织多肽组学的研究方法提供了有价值的科学资料.

This study investigated the proteomic changes at different time points in the precipitated pellets of rat spinal cords after applying complete spinal cord transection. By two-dimensional electrophoresis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, MALDI-TOF/TOF and peptide mass fingerprinting analysis, 44 proteins were identified, most of which are membrane and/or organellar proteins. They are mainly involved in metabolic processes (75%), developmental processes (30%), or responses to stimuli (30%), playing negative or positive roles. In particular, decreases of pyruvate dehydrogenase beta, aconitase 2, fumarate hydratase 1, and ATP synthase subunit 6 can lead to ATP depletion by crippling tricarboxylic acid cycle and oxidative phosphorylation. Decreases of several antioxidant proteins such as catalase, peroxiredoxin 1, Parkinson disease 7, and stress-induced phosphoprotein 1 can contribute to the secondary injury of spinal cord. Decreases of development-related 3-phosphoglycerate dehydrogenase and stathmin 1 may be not propitious for spinal cord regeneration. On the other hand, increases of isocitrate dehydrogenase 3 alpha/gamma and glutamate dehydrogenase 1 can help compensate the impaired energy metabolism. Increases of sirtuin 2, crystallin alpha B (CRYAB), and heat shock 27-kDa protein 1 can help resist stresses induced by injury. Increases of adenylate cyclase-associated protein 1 and galactose binding lectin 3 can help regeneration by replaying their roles in neural development. To our knowledge, this is the first case of characterization of the proteomic changes seen in the precipitated fraction of injured spinal cord. Most of the identified proteins were found for the first time to be differentially expressed after spinal cord injury, which may provide new clues about the molecular mechanisms of spinal cord injury and repair.

Two-dimensional electrophorograms of normal and ASCI rats were established to investigate the pathological and repair mechanisms of acute spinal cord injury(ASCI) from the standpoint of protein integrity level.The results indicated that about 1 170±62 and 1 324±47 protein spots in normal and ASCI rat tissues were detected by ImageMaster analysis software.Sixteen differential protein spots varying by a factor of 3-25 were confirmed,among which 10 were up-regulated and 6 were down-regulated in the ASCI rat group.In addition,16 differential proteins were futher identified by MALDI-TOF/TOF-MS,among which 6 were newly identified,and two were of the same protein,viz.neurofilament medium polypeptide(NF-M),but with different modification.Only myelin basic protein(MBP) and NF-M have been confirmed to be closely related with ASCI.

Objective:The application of nanoelectrospray tandem mass spectrometry in characterize and analysis of the primary structure of modified and unknown polypeptides.is expleined by example of 2 polypeptides.Methods: The molecular weights of two peptides were determined in TOF MS mode.Then fragmentation ions of [M+2H]~(2+) ion extracted from the nanoelectrospray tandem mass spectrometry (MS/MS) were subsequently obtained.Results: The primary structure of triptorelin is determined to be E'HWSYWLRPG',of which the N - terminal is pyroglutamic acid(E') and the C -termianl is glycinamide (G').The primary structure of unknown peptide was identified to be T'VSP~* VWLPPSVY by sequence docking method,of whieh the N -terminal of the peptide,threonine (T')was phos- phorylated and the proline (fouth position) is conjugated with sodium ion (P*).Conclution:The data suggested that electrospray tandem mass spectrometry technique have obvious advantage in analyzing the full sequence of mod- ified or unknown peptides.