蛋白质—配体及蛋白质—细胞相互作用的研究策略
详细信息    本馆镜像全文|  推荐本文 |  |   获取CNKI官网全文
摘要
蛋白质几乎参与所有的生物学过程。他们通过与其他蛋白质或者其他分子发生相互作用行使功能。本论文建立了两个研究蛋白质相互作用的蛋白质组学新策略:4Facts策略用于鉴定实时的蛋白质-配体相互作用;4S策略用于整体分析蛋白质-细胞相互作用。
     4Facts策略——快速固定(Fast Fix)、亲和捕获(Fish)、分子量筛选(Filter),可以在不对生物学样品进行预先干预的条件下全面、灵敏而准确地研究实时原位的蛋白质-配体相互作用。在本策略中,SDS-PAGE (十二烷基硫酸钠聚丙烯酰胺凝胶电泳)被用于打断非共价结合以去除未被共价交联的复合物,以及提供分子量信息用于筛选配体蛋白。候选蛋白被认定为靶蛋白的配体蛋白,需要同时满足以下三个条件:第一,必须与靶蛋白在同一胶条中被鉴定;第二,必须在实验组胶条中被鉴定,而不出现在相同、相邻或更高分子量范围的对照组胶条中;第三,所在胶条的分子量范围包含或大于它与靶蛋白的理论分子量之和。我们将4Facts策略应用于人血液中的蛋白质-配体相互作用,以血浆白蛋白以及转铁蛋白为例展示该策略的可行性。人血液不经任何处理被直接加入10%的甲醛溶液中进行5秒的快速交联反应。运用该策略,我们鉴定到35个白蛋白的配体以及52个转铁蛋白的配体。与此前三个主要的白蛋白相互作用蛋白质组学研究进行比较,本研究鉴定到的35个白蛋白配体中的68.57%在其他研究中也被鉴定到。该策略实现了5秒的短时间交联,未来可能被应用于研究动态的蛋白质-蛋白质相互作用。
     4S策略——表面酶切(Surface Shaving)结合种属特异性(Species Specificity),可以被用于整体分析不同物种间的蛋白质-细胞相互作用。我们以牛血清-人Ca Ski细胞相互作用为例来说明4S策略。牛血清培养的Ca Ski细胞在经过三次PBS洗涤之后与胰酶孵育。表面酶切上清以及作为对照的牛血清和细胞裂解液被收集,用于质谱分析。一些蛋白质因为共享多肽而无法被定量。我们把共享多肽的谱图数按照各蛋白所含种属特异的多肽的谱图数的比例,分配给各个蛋白,然后通过NSAF值对牛源蛋白(包含牛种属特异的肽)以及人源蛋白(包含人种属特异的肽)进行定量。运用该策略,30个牛源蛋白相对于牛血清被富集,我们认为这些牛血清蛋白与人Ca Ski细胞发生相互作用,可能在细胞培养中起着重要作用。基因注释显示它们之中的大部分具有结合功能。另外,216个人源蛋白相对于细胞裂解液被富集,它们可能是细胞膜蛋白或者分泌蛋白。该策略实现了对不同物种间蛋白质-细胞相互作用的整体分析,未来可能被应用于病原-宿主微环境相互作用。
Proteins mediate virtually all biological process. They function through interactions with other proteins as well as other molecules. In this dissertation, studies have been made on setting up new proteomic strategies for identification of real-time protein-ligand interactions and for global analyses of protein-cell interactions.
     The4Facts strategy-Fast Fix, Fish, and Filter, can be used to comprehensively, sensitively and precisely identify real-time protein-protein interactions without prior intervention. In this strategy, SDS-PAGE is used to disrupt non-covalent bonds, thereby eliminating uncross-linked complexes and simultaneously providing molecular weight information for identification. Only proteins that simultaneously satisfy the following three criteria can be considered true ligands:1. the target protein must be identified in the same slice as its ligands;2. the ligands must be identified in slices for the experimental group but not in the corresponding, neighboring or higher control slices;3. the ligands appear in the range of molecular weights equal to or greater than the sum of the theoretical molecular weights of the target and the ligand. Using albumin and transferrin as examples, the4Facts strategy was applied to identify protein-ligand interactions in human blood. Human blood was directly added to10%formaldehyde for5s of cross-linking. The use of this strategy identified35ligands for albumin and52ligands for transferrin. Comparison with three major previous studies of the albuminome revealed that68.57%of the35albumin ligands identified in our study were also identified in these other studies. Since fast fixing of5s was achieved, this strategy can be potentially used in identification of dynamic protein-protein interactions, and become a new standard in future.
     The4S strategy-Surface Shaving and Species Specificity, can be used to achieve global analyses of protein-cell interactions between different species. Bovine serum-human Ca Ski cell interaction was used as an example to illustrate this strategy. Cells cultured in bovine serum were washed three times with PBS and then incubated with trypsin. The supernatant after cell surface shaving were send to MS analysis. Bovine serum and Ca Ski cell lysate were analyzed as control. Many proteins were reported in protein groups and cannot be quantified because of shared peptides. We assigned SpC (spectral counts) of shared peptides to different proteins according to the SpC ratio of species specific peptides in those proteins. Bovine proteins (proteins with bovine specific peptides) and human proteins (proteins with human specific peptides) were quantified by spectral counting using NSAF values. Using this strategy,30bovine proteins were identified as proteins that interact with Ca Ski cells as they were enriched comparing with bovine serum. They might play important roles in cell culture. Most of them have binding function with GO annotation. In addition,216human proteins were enriched comparing with cell lysate. They might be cell membrane proteins or secreted proteins. This strategy achieved global analyses of protein-cell interactions between different species, and can be potentially used in identification of pathogen-host microenvironment interactions, etc.
引文
[1]. Klockenbusch, C.; Kast, J., Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin betal. [J]. J Biomed Biotechnol 2010,2010, 927585.
    [2]. Klockenbusch, C.; O'Hara, J. E.; Kast, J., Advancing formaldehyde cross-linking towards quantitative proteomic applications. [J]. Anal Bioanal Chem 2012,404, (4), 1057-67.
    [3]. Sutherland, B. W.; Toews, J.; Kast, J., Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions. [J]. J Mass Spectrom 2008, 43,(6),699-715.
    [4]. Toews, J.; Rogalski, J. C.; Clark, T. J.; Kast, J., Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions. [J]. Anal Chim Acta 2008,618, (2),168-83.
    [5]. Vasilescu, J.; Guo, X.; Kast, J., Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry. [J]. Proleomics 2004,4, (12),3845-54.
    [6]. Tjalsma, H.; Lambooy, L.; Hermans, P. W.; Swinkels, D. W., Shedding & shaving: disclosure of proteomic expressions on a bacterial face. [J]. Proteomics 2008,8, (7), 1415-28.
    [7]. Solis, N.; Larsen, M. R.; Cordwell, S. J., Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false-positive control. [J]. Proteomics 2010, 10, (10),2037-49.
    [8]. Bjorke, H.; Andersson, K., Measuring the affinity of a radioligand with its receptor using a rotating cell dish with in situ reference area. [J]. Appl Radial Isot 2006,64, (1), 32-7.
    [9]. Bjorke, H.; Andersson, K., Automated, high-resolution cellular retention and uptake studies in vitro. [J]. Appl Radiat Isot 2006,64, (8),901-5.
    [10]. Dreisbach, A.; van der Kooi-Pol, M. M.; Otto, A.; Gronau, K.; Bonarius, H. P.; Westra, H.; Groen, H.; Becher, D.; Hecker, M.; van Dijl, J. M., Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface. [J]. Proteomics 2011,11,(14),2921-30.
    [11]. Chen, Y.; Gu, B.; Wu, S.; Sun, W.; Ma, S.; Liu, Y.; Gao, Y., Using enrichment index for quality control of secretory protein sample and identification of secretory proteins. [J]. J Mass Spectrom 2009,44, (3),397-403.
    [12]. Zybailov, B. L.; Florens, L.; Washburn, M. P., Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors. [J]. Mol Biosyst 2007,3, (5),354-60.
    [13]. Berggard, T.; Thelin, N.; Falkenberg, C.; Enghild, J. J.; Akerstrom, B., Prothrombin, albumin and immunoglobulin A form covalent complexes with alpha 1-microglobulin in human plasma. [J]. Eur J Biochem 1997,245, (3),676-83.
    [14]. Falkenberg, C.;Enghild, J. J.; Thogersen, I. B.; Salvesen, G.;Akerstrom, B., Isolation and characterization of fibronectin-alpha 1-microglobulin complex in rat plasma. [J]. Biochem J 1994,301 (Pt 3),745-51.
    [15]. Farrah, T.; Deutsch, E. W.; Omenn, G. S.; Campbell, D. S.; Sun, Z.; Bletz, J. A.; Mallick, P.; Katz, J. E.; Malmstrom, J.; Ossola, R.; Watts, J. D.; Lin, B.; Zhang, H.; Moritz, R. L.; Aebersold, R., A high-confidence human plasma proteome reference set with estimated concentrations in PeptideAtlas. [J]. Mol Cell Proteomics 2011,10, (9), Ml 10006353.
    [16]. Gundry, R. L.; White, M. Y.; Nogee, J.; Tchernyshyov, I.; Van Eyk, J. E., Assessment of albumin removal from an immunoaffinity spin column:critical implications for proteomic examination of the albuminome and albumin-depleted samples. [J]. Proteomics 2009,9, (7),2021-8.
    [17]. Camaggi, C. M.; Zavatto, E.; Gramantieri, L.; Camaggi, V.; Strocchi, E.; Righini, R.; Merina, L.; Chieco, P.; Bolondi, L., Serum albumin-bound proteomic signature for early detection and staging of hepatocarcinoma:sample variability and data classification. [J]. Clin Chem Lab Med 2010,48, (9),1319-26.
    [18]. Zhou, M.; Lucas, D. A.; Chan, K. C.; Issaq, H. J.; Petricoin, E. F.,3rd; Liotta, L. A.; Veenstra, T. D.; Conrads, T. P., An investigation into the human serum "interactome". [J]. Electrophoresis 2004,25, (9),1289-98.
    [19]. Gundry, R. L.; Fu, Q.; Jelinek, C. A.; Van Eyk, J. E.; Cotter, R. J., Investigation of an albumin-enriched fraction of human serum and its albuminome. [J]. Proteomics Clin Appl 2007,1,(1),73-88.
    [20]. Scumaci, D.; Gaspari, M.; Saccomanno, M.; Argiro, G.; Quaresima, B.; Faniello, C. M.; Ricci, P.; Costanzo, F.; Cuda, G., Assessment of an ad hoc procedure for isolation and characterization of human albuminome. [J]. Anal Biochem 2011,418, (1),161-3.
    [211. Holewinski, R. J.; Jin, Z.; Powell, M. J.; Maust, M. D.; Van Eyk, J. E., A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid. [J]. Proteomics 2013,13, (5),743-50.
    [22]. Wright, P. C.; Jaffe, S.; Noirel, J.; Zou, X., Opportunities for protein interaction network-guided cellular engineering. [J]. IUBMB Life 2013,65, (1),17-27.
    [23]. Fields, S.; Song, O., A novel genetic system to detect protein-protein interactions. [J]. Nature 1989,340, (6230),245-6.
    [241. Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Seraphin, B., A generic protein purification method for protein complex characterization and proteome exploration. [J]. Nat Biotechnol 1999,17, (10),1030-2.
    [25]. Puig, O.; Caspary, F.; Rigaut, G.; Rutz, B.; Bouveret, E.; Bragado-Nilsson, E.; Wilm, M.; Seraphin, B., The tandem affinity purification (TAP) method:a general procedure of protein complex purification. [J]. Methods 2001,24, (3),218-29.
    [26]. Soderberg, O.; Gullberg, M.; Jarvius, M.; Ridderstrale, K.; Leuchowius, K. J.; Jarvius, J.; Wester, K.; Hydbring, P.; Bahram, F.; Larsson, L. G.; Landegren, U., Direct observation of individual endogenous protein complexes in situ by proximity ligation. [J]. Nat Methods 2006,3, (12),995-1000.
    [27]. Sinz, A., Investigation of protein-protein interactions in living cells by chemical crosslinking and mass spectrometry. [J]. Anal Bioanal Chem 2010,397, (8),3433-40.
    [28]. Clegg, C.; Hayes, D., Identification of neighbouring proteins in the ribosomes of Escherichia coli. A topographical study with the cross-linking reagent dimethyl suberimidate. [J]. Eur J Biochem 1974,42, (1),21-8.
    [29]. Hermanson, G. T., Bioconjugate Techniques,2nd ed. [J]. Elsevier Inc.2008,241.
    [30]. Suchanek, M.; Radzikowska, A.; Thiele, C., Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. [J]. Nat Methods 2005,2, (4),261-7.
    [31]. Xie, J.; Schultz, P. G., A chemical toolkit for proteins-an expanded genetic code. [J]. Nat Rev Mol Cell Biol 2006,7, (10),775-82.
    [32]. Ryu, Y.; Schultz, P. G., Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. [J]. Nat Methods 2006,3, (4),263-5.
    [33]. Tang, X.; Munske, G. R.; Siems, W. F.; Bruce, J. E., Mass spectrometry identifiable cross-linking strategy for studying protein-protein interactions. [J]. Anal Chem 2005,77, (1),311-8.
    [34]. Zhang, H.; Tang, X.; Munske, G. R.; Zakharova, N.; Yang, L.; Zheng, C.; Wolff, M. A.; Tolic, N.; Anderson, G. A.; Shi, L.; Marshall, M. J.; Fredrickson, J. K.; Bruce, J. E., In vivo identification of the outer membrane protein OmcA-MtrC interaction network in Shewanella oneidensis MR-1 cells using novel hydrophobic chemical cross-linkers. [J]. J Proteome Res 2008,7, (4),1712-20.
    [35]. Zhang, H.; Tang, X.; Munske, G. R.; Tolic, N.; Anderson, G. A.; Bruce, J. E., Identification of protein-protein interactions and topologies in living cells with chemical cross-linking and mass spectrometry. [J]. Mol Cell Proteomics 2009,8, (3),409-20.
    [36]. Yang, L.; Zheng, C.; Weisbrod, C. R.; Tang, X.; Munske, G. R.; Hoopmann, M. R.; Eng, J. K.; Bruce, J. E., In vivo application of photocleavable protein interaction reporter technology.[J].J Proteome Res 2012,11, (2),1027-41.
    [37]. Yang, L.; Tang, X.; Weisbrod, C. R.; Munske, G. R.; Eng, J. K.; von Haller, P. D.; Kaiser, N. K.; Bruce, J. E., A photocleavable and mass spectrometry identifiable cross-linker for protein interaction studies. [J]. Anal Chem 2010,82, (9),3556-66.
    [38]. Guerrero, C.; Tagwerker, C.; Kaiser, P.; Huang, L., An integrated mass spectrometry-based proteomic approach:quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. [J]. Mol Cell Proteomics 2006,5, (2),366-78.
    [39]. Hood, B. L.; Darfler, M. M.; Guiel, T. G.; Furusato, B.; Lucas, D. A.; Ringeisen, B. R.; Sesterhenn, I. A.; Conrads, T. P.; Veenstra, T. D.; Krizman, D. B., Proteomic analysis of formalin-fixed prostate cancer tissue. [J]. Mol Cell Proteomics 2005,4, (II), 1741-53.
    [401. Patel, V.; Hood, B. L.; Molinolo, A. A.; Lee, N. H.; Conrads, T. P.; Braisted, J. C. Krizman, D. B.; Veenstra, T. D.; Gutkind, J. S., Proteomic analysis of laser-captured paraffin-embedded tissues:a molecular portrait of head and neck cancer progression. [J]. Clin Cancer Res 2008,14, (4),1002-14.
    [41]. Skare, J. T.; Ahmer, B. M.; Seachord, C. L.; Darveau, R. P.; Postle, K., Energy transduction between membranes. TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA. [J]. J Biol Chem 1993,268,(22),16302-8.
    [42]. Higgs, P. I.; Myers, P. S.; Postle, K., Interactions in the TonB-dependent energy transduction complex:ExbB and ExbD form homomultimers. [J]. J Bacteriol 1998,180, (22),6031-8.
    [43]. Layh-Schmitt, G.; Podtelejnikov, A.; Mann, M., Proteins complexed to the P1 adhesin of Mycoplasma pneumoniae. [J]. Microbiology 2000,146 (Pt 3),741-7.
    [44]. Metz, B.; Kersten, G. F.; Hoogerhout, P.; Brugghe, H. F.; Timmermans, H. A.; de Jong, A.; Meiring, H.; ten Hove, J.; Hennink, W. E.; Crommelin, D. J.; Jiskoot, W., Identification of formaldehyde-induced modifications in proteins:reactions with model peptides. [J].J Biol Chem 2004,279, (8),6235-43.
    [45]. Metz, B.; Kersten, G. F.; Baart, G. J.; de Jong, A.; Meiring, H.; ten Hove, J.; van Steenbergen, M. J.; Hennink, W. E.; Crommelin, D. J.; Jiskoot, W., Identification of formaldehyde-induced modifications in proteins:reactions with insulin. [J]. Bioconjug Chem 2006,17,(3),815-22.
    [46]. Hall, D. B.; Struhl, K., The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo. [J]. J Biol Chem 2002,277, (48),46043-50.
    [47]. Wiczer, B. M.; Bernlohr, D. A., A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes. [J]. J Lipid Res 2009,50, (12),2502-13.
    [48]. Byrum, S.; Mackintosh, S. G.; Edmondson, R. D.; Cheung, W. L.; Taverna, S. D.; Tackett, A. J., Analysis of Histone Exchange during Chromatin Purification. [J]. J Integr OMICS 2011,1,(1),61-65.
    [491. Hwang, S. I.; Thumar, J.; Lundgren, D. H.; Rezaul, K.; Mayya, V.; Wu, L.; Eng, J.; Wright, M. E.; Han, D. K., Direct cancer tissue proteomics:a method to identify candidate cancer biomarkers from formalin-fixed paraffin-embedded archival tissues. [J]. Oncogene 2007,26, (1),65-76.
    [501. Bagnato, C.; Thumar, J.; Mayya, V.; Hwang, S. I.; Zebroski, H.; Claffey, K. P.; Haudenschild, C.; Eng, J. K.; Lundgren, D. H.; Han, D. K., Proteomics analysis of human coronary atherosclerotic plaque:a feasibility study of direct tissue proteomics by liquid chromatography and tandem mass spectrometry. [J]. Mol Cell Proteomics 2007,6, (6),1088-102.
    [51]. Schmitt-Ulms, G.; Hansen, K.; Liu, J.; Cowdrey, C.; Yang, J.; DeArmond, S. J.; Cohen, F. E.; Prusiner, S. B.; Baldwin, M. A., Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues. [J]. Nat Biotechnol 2004,22, (6),724-31.
    [52]. Bousquet-Dubouch, M. P.; Baudelet, E.; Guerin, F.; Matondo, M.; Uttenweiler-Joseph, S.; Burlet-Schiltz, O.; Monsarrat, B., Affinity purification strategy to capture human endogenous proteasome complexes diversity and to identify proteasome-interacting proteins. [J]. Mol Cell Proteomics 2009,8, (5),1150-64.
    [531. Tagwerker, C.; Flick, K.; Cui, M.; Guerrero, C.; Dou, Y.; Auer, B.; Baldi, P.; Huang, L.; Kaiser, P., A tandem affinity tag for two-step purification under fully denaturing conditions:application in ubiquitin profiling and protein complex identification combined with in vivocross-linking. [J]. Mol Cell Proteomics 2006,5, (4), 737-48.
    [54]. Chowdhury,S.M.;Shi,L.;Yoon,H.;Ansong,C.;Rommereim,L.M.:Norbeck, A.D.;Auberry,K.J.;Moore,R.J.;Adkjns,J.N.;Heffron,F.;Smith,R.D.,A method for investigating protein-protein interactions related to salmonella typhimurium pathogenesis. [J].J Proleome Res 2009,8,(3),1504-14.
    [55]. Muller,V.S.;Jungblut,P. R.;Meyer,T.F.;Hunke,S.,Membrane-SPINE:an improved method to identify protein-protein interaction partners of membrane proteins in vivo.[J].Proleomics 2011,11,(10),2124-8.
    [56]. Kaake,R.M.:Milenkovic,T.;Przulj,N.;Kaiser, P.;Huang,L.,Characterization of cell cycle specific protein interaction networks of the yeast 26S proteasome complex by the QTAX strategy.[J]. J Proleome Res 2010,9,(4),2016-29.
    [57]. Watts,J.C.;HUO,H.;Bai,Y;Ehsani,S.;Jeon,A.H.;Shi,T.;Dande,N.;Lau.A.; Young,R.;Xu,L.;Carlson,G.A.;Williams,D.;Westaway,D.;Schmitt-Ulms,G, Interactome analyses identify ties of PrP and its mammalian paralogs to oIigomannosidic N-glycans and endoplasmic reticulum-derived chaperones.[J].PLoS Pathog 2009,5, (10),e1000608.
    [58]. Knobbe,C.B.;Reveu,T.J.;Bai,Y:;Chow,V:;Jeon,A.H.;Bohm,C.;Ehsani,S.; Kislinger,T.;Mount,H.T.;Mak,T.W.;St George-Hyslop,P.;Schmitt-Ulms,G.,Choice of biological source material supersedes oxidative stress in its influence on DJ-1 in vivo interactions with Hsp90.[J].J proteome Res 2011,10,(10),4388-404.
    [59]. Okada,H.;Zhang,W.;Peterhoif,C.;Hwang,J.C.;Nix011,R.A.;Ryu,S.H.; Kim,T.W.,Proteomic identincation of Sorting nexin 6 as a negative regulator of BACEl-mediated APP processing.[J].FASEB,2010,24,(8),2783-94.
    [60]. Rappsilber.J.,The beginning of a beautiful friendship:cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes.[J].. J Sruct Biol 2011,173,(3),530-40.
    [61]. Stengel, F.;Aebersold, R.;Robinson, C. V, Joining forces:integrating proteomics alld cross-linking with the mass spectrometry of intact comp]exes.[J].Mol Cell Proteomics 2012,11,(3),R111014027.
    [62]. Panchaud,A.;Singh,P:;Shaffer,S.A.;G00dlett,D.R.,xComb:a cross-ljnked peptide database approach to protein-protein interaction analysis.[J]. J Proteome Res 2010,9,(5),2508-15.
    [63]. Du,X.;ChOWdhury,S.M.;Manes,N.P;Wu,S.;Mayer,M.U.;Adkins,J.N.; Anderson,G A.;Smith,R.D.,Xlink-identiner:an automated data analysis platform for conndent identifications of chemically cross-linked peptides using tandem mass spectrometry.[J]. J Proteome Res 2011,10,(3),923-31.
    [64]. Fritzsche,R.;IhIing,C.H.;Gotze,M.;Sinz,A.,optimizing the enrichlnent of cross-Iinked products for mass spectrometric protein analysis.[J].JRapid Commun Mass Spectrom 2012,26,(6),653-8.
    [65]. Lauber M.A.;Reill y,J.P.,Structural analysis of a prokaryotic ribosome using a novel amidinating cross-linker and mass spectrometry.[J]. J Proteome Res 2011,10,(8), 3604-16.
    [66]. Leitner A.;Reisch],R.;Walzthoeni,T.;Herzog,F.;Bohn,S.;ForsteL F.; Aebersold,R.,Expanding the chemical cross-linking toolbox by the use of multiple proteases and enrichment by size exclusion chromatography.[J].Mol Cell Proteomics 2012,11,(3),M111 014126.

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址:北京市海淀区学院路29号 邮编:100083

电话:办公室:(+86 10)66554848;文献借阅、咨询服务、科技查新:66554700