建立SILAC定量技术并发现CD40受体激活后招募的复合物新组分
|
摘要
细胞内蛋白质相互作用的研究是目前生命科学研究的热点领域，这些研究有助于我们了解蛋白质功能，以及阐明信号通路转导过程，进而揭示生命的奥秘。随着科学技术的进步，我们目前拥有了不少研究蛋白质相互作用的技术方法，但是面对如此复杂而又精密的生命体系，这些技术所能解决的问题还很局限，其中如何研究蛋白质动态的相互作用就是核心问题之一。同静态的相互作用相比，蛋白质之间的解离、聚合、再解离、再聚合，更吸引着生物学家的目光，因为这往往意味着某条信号通路的激活或抑制，而这些信号通路通常又是引起细胞生长、分化、突变、衰老、死亡等复杂生理或病理变化的始动因素。
肿瘤坏死因子受体超家族中的许多成员在免疫反应中起着至关重要的作用，其中CD40是I型由免疫系统的抗原呈递细胞表达的膜蛋白，是由激活的T细胞表达的细胞表面蛋白CD154的受体，它是MAPK、NFκB通路及非经典NFκB通路的重要激活子。其介导的信号通路不仅对免疫应答至关重要，而且与自身免疫性疾病，神经退行性疾病等密切相关。因此，深入理解CD40信号通路的分子机制以逐渐成为目前研究的热点之一。
目前的研究揭示CD40受体激活的过程显示：CD40首先招募直接与其相互作用的TNF受体相关因子TRAFs（TNF receptor associate factor），并由TRAFs作为adaptor招募下游信号激活关键分子如NFκB和应激激活蛋白激酶等，形成受体复合体；再通过受体复合体中的部分分子激活下游信号转导通路，以实现其生物学效应。但是，CD40在抗原呈递细胞中的传导激活信号机制并不是完全清楚，因此，研究CD40信号复合体的组成及功能将有助于阐明CD40引起的信号转导通路的分子机制，而CD40信号激活状态下的动态蛋白质复合物组成也将成为CD40信号通路研究的前沿
本研究利用最新蛋白质组学定量技术，研究细胞表面受体CD40激活后细胞内的蛋白质相互作用，初步建立一套可用来分析蛋白质动态相互作用的技术体系，并在CD40信号激活过程中，研究寻找CD40受体复合物的动态新组成。
蛋白质复合物的研究方法主要包括酵母双杂交、蛋白芯片，串联亲和纯化结合质谱法以及基于生物信息学的分析等。近年来，基于质谱的定量蛋白质组学技术发展日新月异，又为生物学家研究蛋白质相互作用提供了一种新的技术手段。基于质谱的定量蛋白质组学技术应用于相互作用的研究原理是用亲和纯化的方式富集对照和实验组蛋白复合体，其中，背景蛋白和非特异结合是等量存在的，当实验组信号通路激活时，实验组结合的特异蛋白质的量与对照组相比会发生变化（上调或者下调）。从而可以排除背景和非特异结合蛋白，识别与信号转导通路相关的特异性结合蛋白。
这种基于质谱的定量蛋白质组学技术主要包括稳定同位素标记和非标记的定量分析方法，其中，基于稳定同位素标记的方法常用的又分为两类：一类是体外化学标记的蛋白质组定量分析，包括同量异构标记的相对和绝对蛋白定量技术（Isobaric Tagging for Relative and Absolute protein Quantitation, iTRAQ）和同位素亲和标签（Isotope-Coded Affinity Tagging ,ICAT）等，另一类是稳定同位素代谢标记的蛋白质定量分析方法，如细胞培养中氨基酸稳定同位素标记方法（StableIsotope Labeling of Amino acids in Cell Culture,SILAC）。
SILAC技术由于标记效率高、重复性好、变异系数小和定量结果准确度高等特点成为一种分析动态相互作用结合蛋白的最有效的方法。其基本原理是分别利用天然和重型稳定同位素标记的氨基酸培养细胞，对细胞分别进行培养，经5-6个倍增周期后，稳定同位素标记的氨基酸完全掺入到细胞新合成的蛋白质中。轻、重稳定同位素标记的细胞，按细胞数或蛋白量等比例混合后，按蛋白质组学方法制样后，用质谱进行定性和定量分析。SILAC技术用于解析蛋白质相互作用，可以克服传统研究方法（如酵母双杂交、蛋白芯片等）的局限，它可以研究内源性蛋白质相互作用，能有效区别背景与真实的相互作用蛋白质，找出动态结合蛋白质，检测弱的或不稳定的相互作用蛋白质，并能大规模的实时分析蛋白相互作用的动态变化。
本研究根据配体激活CD40激活点实验，选择研究CD40配体刺激10分钟的动态相互作用蛋白，实验选用了传统的SILAC实验步骤即PAM（Purification afterMixing）和改进的SILAC实验步骤MAP（Mixing after Purification）来完成实验，在三次试验中共鉴定出2130个蛋白质，其中310个经软件分析得到定量信息，234个蛋白质在三次实验中均被鉴定出来。配基刺激A20细胞10分钟后上调蛋白质（RATIO>1.3%）51个，下调蛋白质（RATIO<0.7%）14个，在鉴定的蛋白中找到了一些可能参与CD40信号通路的蛋白，其中与泛素蛋白酶体通路相关的有OTUB1、NEDD4，UBR2等；与通路相关的激酶，如MAPKK3和Dual specificitytyrosine phosphorylation regulated kinase 1；与NFκB信号通路相关的蛋白有NFκBp100，NFκB activator 1，Myb binding protein 1，Sequestosome 1等，还有一些具有其他重要功能蛋白有NEDD1，ADPATPtransfor 1，Non POU domaincontaining octamer binding protein等，现在我们正在对上述蛋白进行进一步验证以及功能探索，通过Westen blot我们已经验证了NEDD4与CD40的相互作用，为CD40信号通路的调控及其机制的研究提供了十分重要的线索。
本研究建立了用SILAC定量技术体系研究细胞内CD40激活后蛋白质相互作用的动态变化。不仅为CD40信号通路的研究提供了重要的线索，更为重要的是我们初步搭建了一个研究蛋白质复合物动态变化的技术平台，为其它重要信号通路的研究提供了一个可选的技术手段。
Study of intracellular protein interaction is a hot area of life science.It helps us tounderstand protein function, clarify signaling pathway, then revealed the secret of life.With the advances in science and technology, there are a lot of technologies toresearch protein interaction, but they still have limitations.Therefore, how to study thedynamics interactions of protein is one of the core issues. Compared with the staticinteractions, dynamic protein interactions attract concerns of biologists.
Many tumor necrosis factor receptor superfamily members play a vital role in theimmune response, among them,CD40 is an important promoter in MAPK,NFκB andnon- canonical NFκB pathway. CD40 mediated signaling pathway is essential notonly for the immune response, but also to autoimmune diseases, neurodegenerativediseases, cancer,et.al. Therefore, understanding of molecular mechanisms of CD40signaling pathways has become a research hotspot.
The current study reveals the process of CD40 activation. First, CD40 recruitTNF receptor associated factors TRAFs directly. Second, TRAFs as adaptors,recruit molecules which are essential for downstream signal activation to form areceptor complex. Finally., some molecules of receptor complex activate downstreamsignaling pathway to exert CD40's biological effects. But CD40 on antigen-presentingcells activating signal mechanism is not entirely clear, Therefore, the study of CD40signaling complex composition and function will help to reveal molecular mechanismof transduction and modulation of CD40 signaling.
The purpose of this paper is to use the latest technology of quantitativeproteomics to establish a set of technological system for studying dynamic proteininteraction,and identify the dynamic new composition of CD40 signal complexrecruited upon signal activation.
Research methods of protein complex mainly include yeast two-hybrid, proteinchips, tandem affinity purification combined with mass spectrometry andbioinformatics-based analysis and other methods. In recent years, massspectrometry-based quantitative Proteomics technologies are developing rapidly,provides a new and reliable technologies for biologists to study protein interaction.The principle of these technologies is to enrich protein complex from control groupand experment group of protein, in which the background protein and non-specificprotein is equivalent exists, and the amount of specific complex components willchange(up or down) upon experimental treatment. So the background andnon-specific binding protein will be excluded, and the specific binding proteins willbe recognized.
Quantitative proteomic technology based on Mass spectrometry include stableisotope labeling and lable free quantitative analysis method. The method of based onstable isotope labeling is divided into two class: one is chemical labeling technology,including Isobaric Tagging for Relative and Absolute protein Quantitation(iTRAQ)and Isotope-Coded Affinity Tagging( ICAT), another one is metabolic labelingtechnology. Such as Stable isotope labeling by amino acid in cell culture(SILAC).
With characteristics such as high labeling efficiency, good reproducibility, smallcoefficient of variation and accurate quantitative results, SILAC is becoming a mosteffective way to analyse dynamic protein interactions. Its basic principle is using toincorporate stable isotope into cellular proteomes by adding isotope labeled aminoacids to growth medium-for quantitative analysis. For the analysis of proteininteractions, SILAC technology can overcome the limitations of traditional researchmethod such as yeast two hybrid, protein chip, etc., SILAC can further study theinteraction between endogenous proteins, discriminate effectively the background and real proteins interactions, identify dynamic binding protein, detected a weak orunstable protein interactions, and real-time analysis large-scale dynamic changes inprotein interactions
We have Established SILAC quantitative technologies and used it identify newdynamic binding protein in CD40 signaling complex induced by CD40 activation. Weselected traditional SILAC experimental steps( PAM) and improved SILACexperimental steps(MAP), In three experiments, a total of 2,130 proteins wereidentified, among them 234 proteins were identified in all experiments, 310 proteinswewe quantified, 51 Up Regulated proteins (RATIO>1.3%)51proteins, 14 downRegulated proteins (RATIO<0.7%), In these proteins,some were reported to beinvolved in CD40 signal transcription pathway, such as MAPKK1, MAPKK3,TRAF2 and TRAF3, NFκBp100, NFκB Activator 1,Myb binding protein 1,Ubiquitin protease pathway related protein such as OTUB1, and NEDD4, also somehas other important function protein such as NEDD1, Sequestosome 1 and so on.
Now proteins mentioned above are undergoing further validation as well as thefunction exploration. With Westen-blot, We verified the interaction between NEDD4and CD40, and provided an important clue for the CD40 signaling pathway.
This study establishes a quantitative technology using SILAC for research ofCD40 signal complex, and identifies dynamic interaction of proteins in CD40signaling pathway activation, Not only provides key clues for the CD40 signalingpathway, more importantly, constructs preliminary a technology platform for studyingdynamic protein complex, and also offers a technological choice for other importantsignaling pathway.
肿瘤坏死因子受体超家族中的许多成员在免疫反应中起着至关重要的作用，其中CD40是I型由免疫系统的抗原呈递细胞表达的膜蛋白，是由激活的T细胞表达的细胞表面蛋白CD154的受体，它是MAPK、NFκB通路及非经典NFκB通路的重要激活子。其介导的信号通路不仅对免疫应答至关重要，而且与自身免疫性疾病，神经退行性疾病等密切相关。因此，深入理解CD40信号通路的分子机制以逐渐成为目前研究的热点之一。
目前的研究揭示CD40受体激活的过程显示：CD40首先招募直接与其相互作用的TNF受体相关因子TRAFs（TNF receptor associate factor），并由TRAFs作为adaptor招募下游信号激活关键分子如NFκB和应激激活蛋白激酶等，形成受体复合体；再通过受体复合体中的部分分子激活下游信号转导通路，以实现其生物学效应。但是，CD40在抗原呈递细胞中的传导激活信号机制并不是完全清楚，因此，研究CD40信号复合体的组成及功能将有助于阐明CD40引起的信号转导通路的分子机制，而CD40信号激活状态下的动态蛋白质复合物组成也将成为CD40信号通路研究的前沿
本研究利用最新蛋白质组学定量技术，研究细胞表面受体CD40激活后细胞内的蛋白质相互作用，初步建立一套可用来分析蛋白质动态相互作用的技术体系，并在CD40信号激活过程中，研究寻找CD40受体复合物的动态新组成。
蛋白质复合物的研究方法主要包括酵母双杂交、蛋白芯片，串联亲和纯化结合质谱法以及基于生物信息学的分析等。近年来，基于质谱的定量蛋白质组学技术发展日新月异，又为生物学家研究蛋白质相互作用提供了一种新的技术手段。基于质谱的定量蛋白质组学技术应用于相互作用的研究原理是用亲和纯化的方式富集对照和实验组蛋白复合体，其中，背景蛋白和非特异结合是等量存在的，当实验组信号通路激活时，实验组结合的特异蛋白质的量与对照组相比会发生变化（上调或者下调）。从而可以排除背景和非特异结合蛋白，识别与信号转导通路相关的特异性结合蛋白。
这种基于质谱的定量蛋白质组学技术主要包括稳定同位素标记和非标记的定量分析方法，其中，基于稳定同位素标记的方法常用的又分为两类：一类是体外化学标记的蛋白质组定量分析，包括同量异构标记的相对和绝对蛋白定量技术（Isobaric Tagging for Relative and Absolute protein Quantitation, iTRAQ）和同位素亲和标签（Isotope-Coded Affinity Tagging ,ICAT）等，另一类是稳定同位素代谢标记的蛋白质定量分析方法，如细胞培养中氨基酸稳定同位素标记方法（StableIsotope Labeling of Amino acids in Cell Culture,SILAC）。
SILAC技术由于标记效率高、重复性好、变异系数小和定量结果准确度高等特点成为一种分析动态相互作用结合蛋白的最有效的方法。其基本原理是分别利用天然和重型稳定同位素标记的氨基酸培养细胞，对细胞分别进行培养，经5-6个倍增周期后，稳定同位素标记的氨基酸完全掺入到细胞新合成的蛋白质中。轻、重稳定同位素标记的细胞，按细胞数或蛋白量等比例混合后，按蛋白质组学方法制样后，用质谱进行定性和定量分析。SILAC技术用于解析蛋白质相互作用，可以克服传统研究方法（如酵母双杂交、蛋白芯片等）的局限，它可以研究内源性蛋白质相互作用，能有效区别背景与真实的相互作用蛋白质，找出动态结合蛋白质，检测弱的或不稳定的相互作用蛋白质，并能大规模的实时分析蛋白相互作用的动态变化。
本研究根据配体激活CD40激活点实验，选择研究CD40配体刺激10分钟的动态相互作用蛋白，实验选用了传统的SILAC实验步骤即PAM（Purification afterMixing）和改进的SILAC实验步骤MAP（Mixing after Purification）来完成实验，在三次试验中共鉴定出2130个蛋白质，其中310个经软件分析得到定量信息，234个蛋白质在三次实验中均被鉴定出来。配基刺激A20细胞10分钟后上调蛋白质（RATIO>1.3%）51个，下调蛋白质（RATIO<0.7%）14个，在鉴定的蛋白中找到了一些可能参与CD40信号通路的蛋白，其中与泛素蛋白酶体通路相关的有OTUB1、NEDD4，UBR2等；与通路相关的激酶，如MAPKK3和Dual specificitytyrosine phosphorylation regulated kinase 1；与NFκB信号通路相关的蛋白有NFκBp100，NFκB activator 1，Myb binding protein 1，Sequestosome 1等，还有一些具有其他重要功能蛋白有NEDD1，ADPATPtransfor 1，Non POU domaincontaining octamer binding protein等，现在我们正在对上述蛋白进行进一步验证以及功能探索，通过Westen blot我们已经验证了NEDD4与CD40的相互作用，为CD40信号通路的调控及其机制的研究提供了十分重要的线索。
本研究建立了用SILAC定量技术体系研究细胞内CD40激活后蛋白质相互作用的动态变化。不仅为CD40信号通路的研究提供了重要的线索，更为重要的是我们初步搭建了一个研究蛋白质复合物动态变化的技术平台，为其它重要信号通路的研究提供了一个可选的技术手段。
Study of intracellular protein interaction is a hot area of life science.It helps us tounderstand protein function, clarify signaling pathway, then revealed the secret of life.With the advances in science and technology, there are a lot of technologies toresearch protein interaction, but they still have limitations.Therefore, how to study thedynamics interactions of protein is one of the core issues. Compared with the staticinteractions, dynamic protein interactions attract concerns of biologists.
Many tumor necrosis factor receptor superfamily members play a vital role in theimmune response, among them,CD40 is an important promoter in MAPK,NFκB andnon- canonical NFκB pathway. CD40 mediated signaling pathway is essential notonly for the immune response, but also to autoimmune diseases, neurodegenerativediseases, cancer,et.al. Therefore, understanding of molecular mechanisms of CD40signaling pathways has become a research hotspot.
The current study reveals the process of CD40 activation. First, CD40 recruitTNF receptor associated factors TRAFs directly. Second, TRAFs as adaptors,recruit molecules which are essential for downstream signal activation to form areceptor complex. Finally., some molecules of receptor complex activate downstreamsignaling pathway to exert CD40's biological effects. But CD40 on antigen-presentingcells activating signal mechanism is not entirely clear, Therefore, the study of CD40signaling complex composition and function will help to reveal molecular mechanismof transduction and modulation of CD40 signaling.
The purpose of this paper is to use the latest technology of quantitativeproteomics to establish a set of technological system for studying dynamic proteininteraction,and identify the dynamic new composition of CD40 signal complexrecruited upon signal activation.
Research methods of protein complex mainly include yeast two-hybrid, proteinchips, tandem affinity purification combined with mass spectrometry andbioinformatics-based analysis and other methods. In recent years, massspectrometry-based quantitative Proteomics technologies are developing rapidly,provides a new and reliable technologies for biologists to study protein interaction.The principle of these technologies is to enrich protein complex from control groupand experment group of protein, in which the background protein and non-specificprotein is equivalent exists, and the amount of specific complex components willchange(up or down) upon experimental treatment. So the background andnon-specific binding protein will be excluded, and the specific binding proteins willbe recognized.
Quantitative proteomic technology based on Mass spectrometry include stableisotope labeling and lable free quantitative analysis method. The method of based onstable isotope labeling is divided into two class: one is chemical labeling technology,including Isobaric Tagging for Relative and Absolute protein Quantitation(iTRAQ)and Isotope-Coded Affinity Tagging( ICAT), another one is metabolic labelingtechnology. Such as Stable isotope labeling by amino acid in cell culture(SILAC).
With characteristics such as high labeling efficiency, good reproducibility, smallcoefficient of variation and accurate quantitative results, SILAC is becoming a mosteffective way to analyse dynamic protein interactions. Its basic principle is using toincorporate stable isotope into cellular proteomes by adding isotope labeled aminoacids to growth medium-for quantitative analysis. For the analysis of proteininteractions, SILAC technology can overcome the limitations of traditional researchmethod such as yeast two hybrid, protein chip, etc., SILAC can further study theinteraction between endogenous proteins, discriminate effectively the background and real proteins interactions, identify dynamic binding protein, detected a weak orunstable protein interactions, and real-time analysis large-scale dynamic changes inprotein interactions
We have Established SILAC quantitative technologies and used it identify newdynamic binding protein in CD40 signaling complex induced by CD40 activation. Weselected traditional SILAC experimental steps( PAM) and improved SILACexperimental steps(MAP), In three experiments, a total of 2,130 proteins wereidentified, among them 234 proteins were identified in all experiments, 310 proteinswewe quantified, 51 Up Regulated proteins (RATIO>1.3%)51proteins, 14 downRegulated proteins (RATIO<0.7%), In these proteins,some were reported to beinvolved in CD40 signal transcription pathway, such as MAPKK1, MAPKK3,TRAF2 and TRAF3, NFκBp100, NFκB Activator 1,Myb binding protein 1,Ubiquitin protease pathway related protein such as OTUB1, and NEDD4, also somehas other important function protein such as NEDD1, Sequestosome 1 and so on.
Now proteins mentioned above are undergoing further validation as well as thefunction exploration. With Westen-blot, We verified the interaction between NEDD4and CD40, and provided an important clue for the CD40 signaling pathway.
This study establishes a quantitative technology using SILAC for research ofCD40 signal complex, and identifies dynamic interaction of proteins in CD40signaling pathway activation, Not only provides key clues for the CD40 signalingpathway, more importantly, constructs preliminary a technology platform for studyingdynamic protein complex, and also offers a technological choice for other importantsignaling pathway.
引文
（1）Fields,S.and O,Song.1989.Anovel genetic system to detect protein-proteininteractions.Nature 340:245-246
（2）2Suter,B.,D.Auerbach, and I.stagljar.2006.Yeast-based functional genomicsand proteomics technologies:the first 15 years and beyond.Biotechniques40:625-644
（3）Rigant G,Shevchenko,A,Rutz B,et al,A generic protein purification method forprotein complex characterization and proteome exploration.NatBiotechnol,1999,17(10):1030-1032
（4）Krogan,N.J.,G,Cagney.H.Yu,G.zhiong,et al.2006 Global landscape of proteincomplexes in the yeast saccharomyces cerevisiae.Nature 440:637-643
（5）Dengjel,J.,Kratchmarova,I.&Blagoev,B.Receptor tyrosine kinase signaling:aview from quantitative proteomics.Mol.Biosyst.2009;5,1112-1121
（6）Blagoev,B.,Ong,S.E.,Ktatchmarova,I et al,Temporal analysis ofphosphotyrosine-dependent signaling networks by quantitativeproeomics.Nature Biotech.22,1139-1145
（7）PtaceKJ,Devgan G,Michaud G,et al.Global analysis of proteinphosphorylation in yeast.Nature,2005,438(7068):679-684
（8）Gavin,A.C.,P.Aloy,P.Grandi et al,2006 proteome surey reveals modularity ofthe yeast cell machinery, Nature 440:631-636
（9）Lei F,Xiaorong,W,et al. Characterization of Human COP9 signalosomecomplex using affinity purification and mass spectrometry,2008. J ProteomeRes.7(11):4914-4925
（10）Rinner,O.et al.An integrated mass spectrometric and computational frameworkfor the analysis of protein interaction networks.NatureBiotechnol.25.345-352(2007)
（11）Gygi,S.P. et al.Quantitative analysis of complex protein mixtures usingisotope-coded affinity tags.Nature Biotechnol.17.994-999(1999)
（12）Ross,P.L. et al.Multiplexed protein quantitation in Saccharomyces cerevisiaeusing amine-reactive isobaric tagging reagents. Mol.Cell.Proteomics3.1154-1169(2004)
（13）Delphine, P.,Martin,A.J., et al.Quantitative proteomic analysis of proteincomplexes. 2008 Mol.Cell.Proteomics 7.326-246
（14）Ow,S.Y. et al iTRAQ underestimation in simple and complex mixture:”thegood ,the bad and the ugly”.J.Proteome Res.8,5347-5355(2009)
（15）Zhang,Y.et al.A robust error model for iTRAQ quantification reveals divergentdignaling between oncogenic FLT3 mutants in acute myeloidleukemia.Mol.Cell.Proteomics.9, 780-790
（16）Nielsen,M.L et al.Lodoacetamide-induced artifact mimics ubiquitination inmass spectrometry.Nature methods 5,459-460(2008)
（17）Ong,S.E. et al.Stable isotope labeling bu amino acides in cellculture,SILAC,as simple and accutate approach to expression proteomics.2006Mol.Cell.Proteomics 1,376-386
（18）Blagoev,B.et al.A proteomics strategy to elucidate functional protein-proteininteractions applied to EGF signaling.2004 Nature Biotechnol.21.315-318
（19）Mann,M.Functional and quantitative proteomics using SILAC.NatureRev.Mol.Cell Biol.7.952-958(2006)
（20）Everley,P.A. et al. Enhanced analysis of metastatic prostate cance using stableisotopes and high mass accuracy instrumentation.2006 J.ProteomeRes.5.1224-1231
（21）Trinkle-Mulcahy,L. et al.Repo-Man recruits PP1r to chromatin and is essentialfor cell viability ,2006 J.Cell Biol.172,679-692
（22）Kruger,M.et al.SILAC mouse for quantitative proteomics uncovers kindling-3as an essential factor for red blood cell function. Cell 134,353-364(2008)
（23）Geiger,T.,Cox,J.Pawel,O.,Wisniewski,J.R.&Mann,M. Super SILAC mix forquantitative proteomics of human tumor tissue.Nature methods7,383-385(2010)
（24）Ranish,J.A,et al.The study of macromolecular complexes by quantitativeproteomics.2003Nature Genet 33,349-355
（25）Schulze,W.X.,Man,M.A novel proteomic screen for peptide-proteininteractions.2004.J.Biol.Chem.279.10756-10764
（26）Gingras,A.C.,Aebersold,R.&Raught,B. Advances in protein complex analysisusing mass spectrometry.J.physiol.563,11=21(2005)
（27）Ong,S.E, Mann,M., Mass spectrometry-based proteomics turnsquantitative.Nature Chem.boil.1.252-262(2005)
（28）Foster,L.J.et al.Insulin-dependent interactions of proteins with GLUT4reverled through stable isotope labeling by amino acids in cellculture(SILAC).J.Proteome Res.5.64-75(2006)
（29）Blagoev B,Kratchmaroval I,Ong,S.E.,et al. A proteomics strategy to elucidatefunctional protein-protein interactions applied to EGF signaling. NatBiotechnol, 2003,21:315-318
（30）Selbach,M.,Mann,M.,Protein interaction screening by quantitativeimmunoprecipitation combined with knock-down(QUICK).2006 NatureMethods 3,29 Oct 2006
（31）Xiaorong,W.,Lan H.,Identifying Dynamic interactors of protein complexes byquantitative mass spectrometry.Mol.Cellular Proteomics,2008;7(1),46-57
（32）Bishop,G.A.,Moore,C.R.,Cie,P.,Stunz,L.L,et al.TRAF proteins in Cd40signaling.Adv Exp Med Biol 597:131-151
（33）Ferrari, S.; Giliani, S.; Insalaco, A.; Al-Ghonaium, A.; Soresina, A. R.;Loubser, M.; Avanzini, M. A.; Marconi, M.; Badolato, R.; Ugazio, A. G.; Levy,Y.; Catalan, N.; Durandy, A.; Tbakhi, A.; Notarangelo, L. D.; Plebani, A. :Mutations of CD40 gene cause an autosomal recessive form ofimmunodeficiency with hyper IgM. Proc. Nat. Acad. Sci. 98: 12614-12619,2001.
（34）Dadgostar H, Zarnegar B, Hoffmann A, et al. Cooperation of multiplesignaling pathways in CD40-regulated gene expression in B lymphocytes.Proc Natl Acad Sci USA 2002; 99:1497-502
（35）Bruce H.,Roles of TRAF6 in CD40 signaling. Immunol Res.2007;9(1-3):105-114
（36）KuhnéMR, Robbins M, Hambor JE, Mackey MF, Kosaka Y, Nishimura T,Gigley JP, Noelle RJ, Calderhead DM. Assembly and regulation of the CD40receptor complex in human B cells. J Exp Med. 1997 Jul 21;186(2):337-42.
（37）Rowland,S.L., Tremblay,M.M.,Ellison,J.M., et al, Novel mechanism forTNFR-associated factor 6-dependent cd40 signaling, J Immunol(2007)179;4645-4653
（38）Youcun Qian, Zhendong Zhao, Zhengfan Jiang, and Xiaoxia Li Role of NFκBactivator Act1 in CD40-mediated signaling in epithelial cells. Proc Natl AcadSci U S A. 2002 July 9; 99(14): 9386–91.
（39）Ewen Gallagher, Thomas Enzler, Atsushi Matsuzawa, Amy Anzelon-Mills,Dennis Otero, Ryan Holzer, Edith Janssen, Min Gao & Michael KarinKinase MEKK1 is required for CD40-dependent activation of the kinases Jnkand p38, germinal center formation, B cell proliferation and antibodyproduction Nat Immunol 2007 vol 8, 57.
（40）Matsuzawa, A.; Tseng, P.-H.; Vallabhapurapu, S.; Luo, J.-L.; Zhang, W.; Wang,H.; Vignali, D. A. A.; Gallagher, E.; Karin, M. : Essential cytoplasmictranslocation of a cytokine receptor-assembled signaling complex. Science 321:663-668, 2008. Note: Erratum: Science 322: 375 only, 2008.
（41）Andrej, S.,Henrik,T.,et al In-gel digestion for mass spectrometriccharacterization of proteins and proteomes. Nature Protocols,1(6),2856-286
（42）Daniel, S.S.,Katrin,D.,Costel C.D.,Moses,V.C.,and Thomas,A.N.,Stableisotopic labeling by amino acids in cultured primary neurons, Mol.CellularProteomics,2008;7(6),1067-1076
（43）Edelmann MJ,Iphofer A, Akutsu M,et al,Structural basis and specificity ofhuman otubain 1–mediated deubiquitination 2009 Biochem.J.418(2):379-90
（44）Li S,Zheng H,Mao AP,Zhong B,Li Y et al,Regulation of virus-triggeredsignaling by OTUB1-and OTUB2-mediated deubiquitination of TRAF3 andTRAF6.J Biol Chem.2010 Feb 12;285(7):4291-7
（45）Owen,H,R, Elser,M.,Cheung,E.,et al MYBBP1a is a novel repressor ofNF-kappaB.2007 J.Mol.Biol 2007 Feb 23;366(3):725-36
（46）Tsuchiya M,Katagiri N,Kuroda T,et al.Critical role of the mucleolus inactivation of the p53-dependent postmitotic checkpoint. Biochem Biophys ResCommun,2011407(2):378-382
（47）Perrera C,Colombo R,Valsasina B,Carpinelli P,et al. Identification ofMyb-binding protein 1A(MYBBP1A) as a novel substrate for aurora Bkinase.J Bio Chem,2010 16;285(16):11775-85
（48）Jovana Drinjakovic, Hosung Jung, Douglas S, Campbell, E3 Ligase Nedd4Promotes Axon Branching by Downregulating PTEN, Neuron, 2010, 65,341–357.
（49）Baoli Yang, Denise L Gay, Megan K, Nedd4 augments the adaptive immuneresponse by promoting ubiquitin-mediated degradation of Cbl-b in activated Tcells, Nature Immunology, 2008, 9: 1356–1363.
（50）J. Peng, D. Schwartz, J. E. Elias, C. et al, A proteomics approach tounderstanding protein ubiquitination, Nature Biotechnology, 2003, 21(8):921-926.
（51）Timothy J.W.,Susan G. and Marco C., Fc receptor-like A associates withintracellular IgG and IgM but is dispensable for antigen-specific immuneresponses.J.Immunol.2010 185(5):2960-7
（52）Haren L,Remy MH,Bazin I,et al.2006 NEDD1-dependent recruitment of thegamma-tublin ring complex to the centrosome is necessary for centrioleduplication and spindle assembly. J.Cell Biol.172(4):505-15
（53）Luders J,Patel UK,Stearns T(2006),GCP-WD is a gamma-tublin targentingfactor required for centrosomal and chromatin-mediated microtubulenucleation.Nat.Cell 8（2）：137－47
（54）Jung SY,Malovannaya A,Wei J,et al.2005.proteomic analysis of steaby-statenuclear hormone receptor coactivator complexes.Mol.Endocrinol.19(10):2451-65
（55）Dobrzanski P,Pyseck RP,Bravo R(1995) Specific inhibition of RelB/p52transcriptional activity by the C-terminal domain of p100.Oncogene10(5):1003-7
（56）Basak S,Shih VF,Hoffmann A(2008).Generation and activation of multipledimeric transcription factors within the NF-kappaB signaling system.Nol CellBiol 28(10)：3139－50
（57）Nakamura K,Kimple AJ,Siderovski DP,Johnson GL.PB1 domain interaction ofp62/sequestosome 1 and MEKK3 regulates NF-kappaB activation.J BiolChem,2010 15:285(3):2077-89
（58）Kim JY,Ozato K. The sequestosome 1/p62 attenuates cytokine gene expressionin activated macrophages by inhibiting IFN regulatory factor 8 and TNFreceptor-associated factor 6/NF-kappaB activity.2009 15;182(4):2131-40
（59）Hattori M,Minato N，Rap 1 GTPase:functions,regulation,andmalignancy.J.Biochem,134(4):479-84
（60）Mochizuki N,Ohba Y,Kiyokawa E,et al.Activation of the ERK/MAPKpathway by an isoform of rap1GAP associated with G alpha(i) Nature400(6747):891-4
（61）Van Hoof,D., Pinkse,M.W.,Oostwaard,D.W.,Mummery,C.L.,Heck,A.J.,andKrijgsveld,J.(2007) An experimental correctio for arginine-to-prolineconversion artifacts in SILAC-based quantitative proteomics.Nat.Methods4.677-678
（62）Schmidt,F.,Strozynski,M.,Salus,S.s.,Nisen,H.,and Thiede,B. Rapiddetermination of amino acid incorporation by stable isotope labeling withamino acids in cell culture(SILAC).Rapid Commum.MassSpectrom.21,3919-3926
（63）Sean C.Bendall, Chris Hughes,Morag H,Stewart,et al.Prevention of AminoAcid Conversion in SILAC Experiments with Embryonic Stem CellsMol.Cellular Proteomics,2008;7(9),1587-1597
（64）Ong,S.E.,Kratchmarova,I.,and Mann,M., Properties of 13C-substitutedarginine in stable isotope labeling by amino acids in cellculture(SILAC).J,Proteome Res.2,173-181
（1）Fields,S.and O,Song.1989.Anovel genetic system to detect protein-proteininteractions.Nature 340:245-246
（2）2Suter,B.,D.Auerbach, and I.stagljar.2006.Yeast-based functional genomicsand proteomics technologies:the first 15 years and beyond.Biotechniques40:625-644
（3）Rigant G,Shevchenko,A,Rutz B,et al,A generic protein purification method forprotein complex characterization and proteome exploration.NatBiotechnol,1999,17(10):1030-1032
（4）Krogan,N.J.,G,Cagney.H.Yu,G.zhiong,et al.2006 Global landscape of proteincomplexes in the yeast saccharomyces cerevisiae.Nature 440:637-643
（5）Dengjel,J.,Kratchmarova,I.&Blagoev,B.Receptor tyrosine kinase signaling:aview from quantitative proteomics.Mol.Biosyst.2009;5,1112-1121
（6）Blagoev,B.,Ong,S.E.,Ktatchmarova,I et al,Temporal analysis ofphosphotyrosine-dependent signaling networks by quantitativeproeomics.Nature Biotech.22,1139-1145
（7）PtaceKJ,Devgan G,Michaud G,et al.Global analysis of proteinphosphorylation in yeast.Nature,2005,438(7068):679-684
（8）Gavin,A.C.,P.Aloy,P.Grandi et al,2006 proteome surey reveals modularity ofthe yeast cell machinery, Nature 440:631-636
（9）Lei F,Xiaorong,W,et al. Characterization of Human COP9 signalosomecomplex using affinity purification and mass spectrometry,2008. J ProteomeRes.7(11):4914-4925
（10）Rinner,O.et al.An integrated mass spectrometric and computational frameworkfor the analysis of protein interaction networks.NatureBiotechnol.25.345-352(2007)
（11）Andersen，J.S.,Wilkinson,C.J.,Mayor,T.,et al.Proteomic characterizatio of ehthuman centrosome by protein correlatio profiling. Nature 2003,426:570-574
（12）Rinner,O.,Mueller,L.N.,Hubalek,M.,Gstaiger,M. and Aebersold,R., Anintegrated mass spectrometric and computational framewoek for the analysisof protein interaction networks.2007.Nat.Biotechnol.25,345-352
（13）Sutherland,B.W.,Toews,J.,Kast,J.,Utility of formaldehyde cross-linking andmass spectrometry in the study of protein–protein interactions.J MassSpectrom 2008,43(6):699-715
（14）Gygi,S.P. et al.Quantitative analysis of complex protein mixtures usingisotope-coded affinity tags.Nature Biotechnol.17.994-999(1999)
（15）Ross,P.L. et al.Multiplexed protein quantitation in Saccharomyces cerevisiaeusing amine-reactive isobaric tagging reagents. Mol.Cell.Proteomics3.1154-1169(2004)
（16）Delphine, P.,Martin,A.J., et al.Quantitative proteomic analysis of proteincomplexes. 2008 Mol.Cell.Proteomics 7.326-246
（17）Ow,S.Y. et al iTRAQ underestimation in simple and complex mixture:”thegood ,the bad and the ugly”.J.Proteome Res.8,5347-5355(2009)
（18）Zhang,Y.et al.A ribust error model for iTRAQ quantification reveals divergentdignaling between oncogenic FLT3 mutants in acute myeloidleukemia.Mol.Cell.Proteomics 9, 780-790
（19）Ranish,J.A., et al .Identification of TFB5,a new component of generaltranscription and DNA repaire factor IIH.Nature Genet.36,707-713(2004)
（20）Giglia-Mari,G.,et al.A new,tenth submit of TFIIH is responsible for the DNArepair syndrome triothiodystrophy group A.
（21）Zhang,Y.,et al .Time-resolved mass spectrometry of tyrosine phosphorylationsites in the epidermal growth factor receptor signaling network revealsdynamic modules.Mol.Cell.Proteomics 2005,4,1240-1250
（22）Choe,L.,D’Ascenzo,M.，Relkin,N.R.,et al.Eight-plex quantitation of changesin cerebrospinal fluid protein expression in subjects undergoing intravenousimmunoglobulin treatment for Alzhermer’s diease.Proteomics 7,3651-3660.
（23）Nielsen,M.L et al.Lodoacetamide-induced artifact mimics ubiquitination inmass spectrometry.Nature methods 5,459-460(2008)
（24）Suraj Dhungana,B .Alex Merrick,Kenneth B.Tomer.and Michael B.Fessler.Quantitative proteomics analysis of macrophage rafts revealscompartmentalized activation of the proteasome and of proteasome-mediatedERK activation in response tolipopolysaccharide.Mol.Cell.Proteomics.2009,8.1:201-213
（25）Meng Qiao,Yaqu Wang,Xiaoen Xu,et al Mst1 is a interacting protein thatmediates PHLPPs’induced apoptosis.Molecular Cell,2010,38:512-523
（26）Ong,S.E. et al.Stable isotope labeling bu amino acides in cellculture,SILAC,as simple and accutate approach to expression proteomics.2006Mol.Cell.Proteomics 1,376-386
（27）Blagoev,B.et al.A proteomics strategy to elucidate functional protein-proteininteractions applied to EGF signaling.2004 Nature Biotechnol.21.315-318
（28）Mann,M.Functional and quantitative proteomics using SILAC.NatureRev.Mol.Cell Biol.7.952-958(2006)
（29）Everley,P.A. et al. Enhanced analysis of metastatic prostate cance using stableisotopes and high mass accuracy instrumentation.2006 J.ProteomeRes.5.1224-1231
（30）Trinkle-Mulcahy,L. et al.Repo-Man recruits PP1r to chromatin and is essentialfor cell viability ,2006 J.Cell Biol.172,679-692
（31）Kruger,M.et al.SILAC mouse for quantitative proteomics uncovers kindling-3as an essential factor for red blood cell function. Cell 134,353-364(2008)
（32）Geiger,T.,Cox,J.Pawel,O.,Wisniewski,J.R.&Mann,M. Super SILAC mix forquantitative proteomics of human tumor tissue.Nature methods7,383-385(2010)
（33）Ranish,J.A,et al.The study of macromolecular complexes by quantitativeproteomics.2003Nature Genet 33,349-355
（34）Schulze,W.X.,Man,M.A novel proteomic screen for peptide-proteininteractions.2004.J.Biol.Chem.279.10756-10764
（35）Gingras,A.C.,Aebersold,R.&Raught,B. Advances in protein complex analysisusing mass spectrometry.J.physiol.563,11=21(2005)
（36）Ong,S.E, Mann,M., Mass spectrometry-based proteomics turnsquantitative.Nature Chem.boil.1.252-262(2005)
（37）Foster,L.J.et al.Insulin-dependent interactions of proteins with GLUT4reverled through stable isotope labeling by amino acids in cellculture(SILAC).J.Proteome Res.5.64-75(2006)
（38）Blagoev B,Kratchmaroval I,Ong,S.E.,et al. A proteomics strategy to elucidatefunctional protein-protein interactions applied to EGF signaling. NatBiotechnol, 2003,21:315-318
（39）Selbach,M.,Mann,M.,Protein interaction screening by quantitativeimmunoprecipitation combined with knock-down(QUICK).2006 NatureMethods 3,29 Oct 2006
（40）Xiaorong,W.,Lan H.,Identifying Dynamic interactors of protein complexes byquantitative mass spectrometry.Mol.Cellular Proteomics,2008;7(1),46-57
（41）Fan,L.,Wang,X.,Yamoah,K.,et al.Characterization of the human COP9signalosome complex using affinity purification and massspectrometry,J.Proteome Res.&,4914-4925
（42）Mousson F,Kolkman A, Pijnappel W.W.M.P.,et al.Quantitative proteomicsreveals regulation of dynamic components within TATA-binding protein(TBP)transcription complexes.Mol.Cell Proteomics,2008,7:845-852
（43）Seebacher J,Mallick P,Zhang N,Eddes JS,et al.Protein cross-linking analysisusing mass spectrometry,isotope-coded cross-linkers,and integratedcomputational data processing.J Proteome Res 2006,5:2270`-2282
（44）Sinz A.,Chemical cross-linking and mass spectrometry to mapthree-dimensional protein structures and protein-protein interactions.MassSpectrom Rev 2006,25:663-682
（45）Schulze,W.X.,Mann,M.,A novel proteomic screen forpeptide-proteininteractions.J.Biol.Chem.2004,279：10756-10764
（46）Falk,B., Marion,S.,Mario,M.,Mathias M., Unbiased RNA-protein interactionscreen by quantitative proteomics.PNAS,2009,106(26)10626-10631
（2）2Suter,B.,D.Auerbach, and I.stagljar.2006.Yeast-based functional genomicsand proteomics technologies:the first 15 years and beyond.Biotechniques40:625-644
（3）Rigant G,Shevchenko,A,Rutz B,et al,A generic protein purification method forprotein complex characterization and proteome exploration.NatBiotechnol,1999,17(10):1030-1032
（4）Krogan,N.J.,G,Cagney.H.Yu,G.zhiong,et al.2006 Global landscape of proteincomplexes in the yeast saccharomyces cerevisiae.Nature 440:637-643
（5）Dengjel,J.,Kratchmarova,I.&Blagoev,B.Receptor tyrosine kinase signaling:aview from quantitative proteomics.Mol.Biosyst.2009;5,1112-1121
（6）Blagoev,B.,Ong,S.E.,Ktatchmarova,I et al,Temporal analysis ofphosphotyrosine-dependent signaling networks by quantitativeproeomics.Nature Biotech.22,1139-1145
（7）PtaceKJ,Devgan G,Michaud G,et al.Global analysis of proteinphosphorylation in yeast.Nature,2005,438(7068):679-684
（8）Gavin,A.C.,P.Aloy,P.Grandi et al,2006 proteome surey reveals modularity ofthe yeast cell machinery, Nature 440:631-636
（9）Lei F,Xiaorong,W,et al. Characterization of Human COP9 signalosomecomplex using affinity purification and mass spectrometry,2008. J ProteomeRes.7(11):4914-4925
（10）Rinner,O.et al.An integrated mass spectrometric and computational frameworkfor the analysis of protein interaction networks.NatureBiotechnol.25.345-352(2007)
（11）Gygi,S.P. et al.Quantitative analysis of complex protein mixtures usingisotope-coded affinity tags.Nature Biotechnol.17.994-999(1999)
（12）Ross,P.L. et al.Multiplexed protein quantitation in Saccharomyces cerevisiaeusing amine-reactive isobaric tagging reagents. Mol.Cell.Proteomics3.1154-1169(2004)
（13）Delphine, P.,Martin,A.J., et al.Quantitative proteomic analysis of proteincomplexes. 2008 Mol.Cell.Proteomics 7.326-246
（14）Ow,S.Y. et al iTRAQ underestimation in simple and complex mixture:”thegood ,the bad and the ugly”.J.Proteome Res.8,5347-5355(2009)
（15）Zhang,Y.et al.A robust error model for iTRAQ quantification reveals divergentdignaling between oncogenic FLT3 mutants in acute myeloidleukemia.Mol.Cell.Proteomics.9, 780-790
（16）Nielsen,M.L et al.Lodoacetamide-induced artifact mimics ubiquitination inmass spectrometry.Nature methods 5,459-460(2008)
（17）Ong,S.E. et al.Stable isotope labeling bu amino acides in cellculture,SILAC,as simple and accutate approach to expression proteomics.2006Mol.Cell.Proteomics 1,376-386
（18）Blagoev,B.et al.A proteomics strategy to elucidate functional protein-proteininteractions applied to EGF signaling.2004 Nature Biotechnol.21.315-318
（19）Mann,M.Functional and quantitative proteomics using SILAC.NatureRev.Mol.Cell Biol.7.952-958(2006)
（20）Everley,P.A. et al. Enhanced analysis of metastatic prostate cance using stableisotopes and high mass accuracy instrumentation.2006 J.ProteomeRes.5.1224-1231
（21）Trinkle-Mulcahy,L. et al.Repo-Man recruits PP1r to chromatin and is essentialfor cell viability ,2006 J.Cell Biol.172,679-692
（22）Kruger,M.et al.SILAC mouse for quantitative proteomics uncovers kindling-3as an essential factor for red blood cell function. Cell 134,353-364(2008)
（23）Geiger,T.,Cox,J.Pawel,O.,Wisniewski,J.R.&Mann,M. Super SILAC mix forquantitative proteomics of human tumor tissue.Nature methods7,383-385(2010)
（24）Ranish,J.A,et al.The study of macromolecular complexes by quantitativeproteomics.2003Nature Genet 33,349-355
（25）Schulze,W.X.,Man,M.A novel proteomic screen for peptide-proteininteractions.2004.J.Biol.Chem.279.10756-10764
（26）Gingras,A.C.,Aebersold,R.&Raught,B. Advances in protein complex analysisusing mass spectrometry.J.physiol.563,11=21(2005)
（27）Ong,S.E, Mann,M., Mass spectrometry-based proteomics turnsquantitative.Nature Chem.boil.1.252-262(2005)
（28）Foster,L.J.et al.Insulin-dependent interactions of proteins with GLUT4reverled through stable isotope labeling by amino acids in cellculture(SILAC).J.Proteome Res.5.64-75(2006)
（29）Blagoev B,Kratchmaroval I,Ong,S.E.,et al. A proteomics strategy to elucidatefunctional protein-protein interactions applied to EGF signaling. NatBiotechnol, 2003,21:315-318
（30）Selbach,M.,Mann,M.,Protein interaction screening by quantitativeimmunoprecipitation combined with knock-down(QUICK).2006 NatureMethods 3,29 Oct 2006
（31）Xiaorong,W.,Lan H.,Identifying Dynamic interactors of protein complexes byquantitative mass spectrometry.Mol.Cellular Proteomics,2008;7(1),46-57
（32）Bishop,G.A.,Moore,C.R.,Cie,P.,Stunz,L.L,et al.TRAF proteins in Cd40signaling.Adv Exp Med Biol 597:131-151
（33）Ferrari, S.; Giliani, S.; Insalaco, A.; Al-Ghonaium, A.; Soresina, A. R.;Loubser, M.; Avanzini, M. A.; Marconi, M.; Badolato, R.; Ugazio, A. G.; Levy,Y.; Catalan, N.; Durandy, A.; Tbakhi, A.; Notarangelo, L. D.; Plebani, A. :Mutations of CD40 gene cause an autosomal recessive form ofimmunodeficiency with hyper IgM. Proc. Nat. Acad. Sci. 98: 12614-12619,2001.
（34）Dadgostar H, Zarnegar B, Hoffmann A, et al. Cooperation of multiplesignaling pathways in CD40-regulated gene expression in B lymphocytes.Proc Natl Acad Sci USA 2002; 99:1497-502
（35）Bruce H.,Roles of TRAF6 in CD40 signaling. Immunol Res.2007;9(1-3):105-114
（36）KuhnéMR, Robbins M, Hambor JE, Mackey MF, Kosaka Y, Nishimura T,Gigley JP, Noelle RJ, Calderhead DM. Assembly and regulation of the CD40receptor complex in human B cells. J Exp Med. 1997 Jul 21;186(2):337-42.
（37）Rowland,S.L., Tremblay,M.M.,Ellison,J.M., et al, Novel mechanism forTNFR-associated factor 6-dependent cd40 signaling, J Immunol(2007)179;4645-4653
（38）Youcun Qian, Zhendong Zhao, Zhengfan Jiang, and Xiaoxia Li Role of NFκBactivator Act1 in CD40-mediated signaling in epithelial cells. Proc Natl AcadSci U S A. 2002 July 9; 99(14): 9386–91.
（39）Ewen Gallagher, Thomas Enzler, Atsushi Matsuzawa, Amy Anzelon-Mills,Dennis Otero, Ryan Holzer, Edith Janssen, Min Gao & Michael KarinKinase MEKK1 is required for CD40-dependent activation of the kinases Jnkand p38, germinal center formation, B cell proliferation and antibodyproduction Nat Immunol 2007 vol 8, 57.
（40）Matsuzawa, A.; Tseng, P.-H.; Vallabhapurapu, S.; Luo, J.-L.; Zhang, W.; Wang,H.; Vignali, D. A. A.; Gallagher, E.; Karin, M. : Essential cytoplasmictranslocation of a cytokine receptor-assembled signaling complex. Science 321:663-668, 2008. Note: Erratum: Science 322: 375 only, 2008.
（41）Andrej, S.,Henrik,T.,et al In-gel digestion for mass spectrometriccharacterization of proteins and proteomes. Nature Protocols,1(6),2856-286
（42）Daniel, S.S.,Katrin,D.,Costel C.D.,Moses,V.C.,and Thomas,A.N.,Stableisotopic labeling by amino acids in cultured primary neurons, Mol.CellularProteomics,2008;7(6),1067-1076
（43）Edelmann MJ,Iphofer A, Akutsu M,et al,Structural basis and specificity ofhuman otubain 1–mediated deubiquitination 2009 Biochem.J.418(2):379-90
（44）Li S,Zheng H,Mao AP,Zhong B,Li Y et al,Regulation of virus-triggeredsignaling by OTUB1-and OTUB2-mediated deubiquitination of TRAF3 andTRAF6.J Biol Chem.2010 Feb 12;285(7):4291-7
（45）Owen,H,R, Elser,M.,Cheung,E.,et al MYBBP1a is a novel repressor ofNF-kappaB.2007 J.Mol.Biol 2007 Feb 23;366(3):725-36
（46）Tsuchiya M,Katagiri N,Kuroda T,et al.Critical role of the mucleolus inactivation of the p53-dependent postmitotic checkpoint. Biochem Biophys ResCommun,2011407(2):378-382
（47）Perrera C,Colombo R,Valsasina B,Carpinelli P,et al. Identification ofMyb-binding protein 1A(MYBBP1A) as a novel substrate for aurora Bkinase.J Bio Chem,2010 16;285(16):11775-85
（48）Jovana Drinjakovic, Hosung Jung, Douglas S, Campbell, E3 Ligase Nedd4Promotes Axon Branching by Downregulating PTEN, Neuron, 2010, 65,341–357.
（49）Baoli Yang, Denise L Gay, Megan K, Nedd4 augments the adaptive immuneresponse by promoting ubiquitin-mediated degradation of Cbl-b in activated Tcells, Nature Immunology, 2008, 9: 1356–1363.
（50）J. Peng, D. Schwartz, J. E. Elias, C. et al, A proteomics approach tounderstanding protein ubiquitination, Nature Biotechnology, 2003, 21(8):921-926.
（51）Timothy J.W.,Susan G. and Marco C., Fc receptor-like A associates withintracellular IgG and IgM but is dispensable for antigen-specific immuneresponses.J.Immunol.2010 185(5):2960-7
（52）Haren L,Remy MH,Bazin I,et al.2006 NEDD1-dependent recruitment of thegamma-tublin ring complex to the centrosome is necessary for centrioleduplication and spindle assembly. J.Cell Biol.172(4):505-15
（53）Luders J,Patel UK,Stearns T(2006),GCP-WD is a gamma-tublin targentingfactor required for centrosomal and chromatin-mediated microtubulenucleation.Nat.Cell 8（2）：137－47
（54）Jung SY,Malovannaya A,Wei J,et al.2005.proteomic analysis of steaby-statenuclear hormone receptor coactivator complexes.Mol.Endocrinol.19(10):2451-65
（55）Dobrzanski P,Pyseck RP,Bravo R(1995) Specific inhibition of RelB/p52transcriptional activity by the C-terminal domain of p100.Oncogene10(5):1003-7
（56）Basak S,Shih VF,Hoffmann A(2008).Generation and activation of multipledimeric transcription factors within the NF-kappaB signaling system.Nol CellBiol 28(10)：3139－50
（57）Nakamura K,Kimple AJ,Siderovski DP,Johnson GL.PB1 domain interaction ofp62/sequestosome 1 and MEKK3 regulates NF-kappaB activation.J BiolChem,2010 15:285(3):2077-89
（58）Kim JY,Ozato K. The sequestosome 1/p62 attenuates cytokine gene expressionin activated macrophages by inhibiting IFN regulatory factor 8 and TNFreceptor-associated factor 6/NF-kappaB activity.2009 15;182(4):2131-40
（59）Hattori M,Minato N，Rap 1 GTPase:functions,regulation,andmalignancy.J.Biochem,134(4):479-84
（60）Mochizuki N,Ohba Y,Kiyokawa E,et al.Activation of the ERK/MAPKpathway by an isoform of rap1GAP associated with G alpha(i) Nature400(6747):891-4
（61）Van Hoof,D., Pinkse,M.W.,Oostwaard,D.W.,Mummery,C.L.,Heck,A.J.,andKrijgsveld,J.(2007) An experimental correctio for arginine-to-prolineconversion artifacts in SILAC-based quantitative proteomics.Nat.Methods4.677-678
（62）Schmidt,F.,Strozynski,M.,Salus,S.s.,Nisen,H.,and Thiede,B. Rapiddetermination of amino acid incorporation by stable isotope labeling withamino acids in cell culture(SILAC).Rapid Commum.MassSpectrom.21,3919-3926
（63）Sean C.Bendall, Chris Hughes,Morag H,Stewart,et al.Prevention of AminoAcid Conversion in SILAC Experiments with Embryonic Stem CellsMol.Cellular Proteomics,2008;7(9),1587-1597
（64）Ong,S.E.,Kratchmarova,I.,and Mann,M., Properties of 13C-substitutedarginine in stable isotope labeling by amino acids in cellculture(SILAC).J,Proteome Res.2,173-181
（1）Fields,S.and O,Song.1989.Anovel genetic system to detect protein-proteininteractions.Nature 340:245-246
（2）2Suter,B.,D.Auerbach, and I.stagljar.2006.Yeast-based functional genomicsand proteomics technologies:the first 15 years and beyond.Biotechniques40:625-644
（3）Rigant G,Shevchenko,A,Rutz B,et al,A generic protein purification method forprotein complex characterization and proteome exploration.NatBiotechnol,1999,17(10):1030-1032
（4）Krogan,N.J.,G,Cagney.H.Yu,G.zhiong,et al.2006 Global landscape of proteincomplexes in the yeast saccharomyces cerevisiae.Nature 440:637-643
（5）Dengjel,J.,Kratchmarova,I.&Blagoev,B.Receptor tyrosine kinase signaling:aview from quantitative proteomics.Mol.Biosyst.2009;5,1112-1121
（6）Blagoev,B.,Ong,S.E.,Ktatchmarova,I et al,Temporal analysis ofphosphotyrosine-dependent signaling networks by quantitativeproeomics.Nature Biotech.22,1139-1145
（7）PtaceKJ,Devgan G,Michaud G,et al.Global analysis of proteinphosphorylation in yeast.Nature,2005,438(7068):679-684
（8）Gavin,A.C.,P.Aloy,P.Grandi et al,2006 proteome surey reveals modularity ofthe yeast cell machinery, Nature 440:631-636
（9）Lei F,Xiaorong,W,et al. Characterization of Human COP9 signalosomecomplex using affinity purification and mass spectrometry,2008. J ProteomeRes.7(11):4914-4925
（10）Rinner,O.et al.An integrated mass spectrometric and computational frameworkfor the analysis of protein interaction networks.NatureBiotechnol.25.345-352(2007)
（11）Andersen，J.S.,Wilkinson,C.J.,Mayor,T.,et al.Proteomic characterizatio of ehthuman centrosome by protein correlatio profiling. Nature 2003,426:570-574
（12）Rinner,O.,Mueller,L.N.,Hubalek,M.,Gstaiger,M. and Aebersold,R., Anintegrated mass spectrometric and computational framewoek for the analysisof protein interaction networks.2007.Nat.Biotechnol.25,345-352
（13）Sutherland,B.W.,Toews,J.,Kast,J.,Utility of formaldehyde cross-linking andmass spectrometry in the study of protein–protein interactions.J MassSpectrom 2008,43(6):699-715
（14）Gygi,S.P. et al.Quantitative analysis of complex protein mixtures usingisotope-coded affinity tags.Nature Biotechnol.17.994-999(1999)
（15）Ross,P.L. et al.Multiplexed protein quantitation in Saccharomyces cerevisiaeusing amine-reactive isobaric tagging reagents. Mol.Cell.Proteomics3.1154-1169(2004)
（16）Delphine, P.,Martin,A.J., et al.Quantitative proteomic analysis of proteincomplexes. 2008 Mol.Cell.Proteomics 7.326-246
（17）Ow,S.Y. et al iTRAQ underestimation in simple and complex mixture:”thegood ,the bad and the ugly”.J.Proteome Res.8,5347-5355(2009)
（18）Zhang,Y.et al.A ribust error model for iTRAQ quantification reveals divergentdignaling between oncogenic FLT3 mutants in acute myeloidleukemia.Mol.Cell.Proteomics 9, 780-790
（19）Ranish,J.A., et al .Identification of TFB5,a new component of generaltranscription and DNA repaire factor IIH.Nature Genet.36,707-713(2004)
（20）Giglia-Mari,G.,et al.A new,tenth submit of TFIIH is responsible for the DNArepair syndrome triothiodystrophy group A.
（21）Zhang,Y.,et al .Time-resolved mass spectrometry of tyrosine phosphorylationsites in the epidermal growth factor receptor signaling network revealsdynamic modules.Mol.Cell.Proteomics 2005,4,1240-1250
（22）Choe,L.,D’Ascenzo,M.，Relkin,N.R.,et al.Eight-plex quantitation of changesin cerebrospinal fluid protein expression in subjects undergoing intravenousimmunoglobulin treatment for Alzhermer’s diease.Proteomics 7,3651-3660.
（23）Nielsen,M.L et al.Lodoacetamide-induced artifact mimics ubiquitination inmass spectrometry.Nature methods 5,459-460(2008)
（24）Suraj Dhungana,B .Alex Merrick,Kenneth B.Tomer.and Michael B.Fessler.Quantitative proteomics analysis of macrophage rafts revealscompartmentalized activation of the proteasome and of proteasome-mediatedERK activation in response tolipopolysaccharide.Mol.Cell.Proteomics.2009,8.1:201-213
（25）Meng Qiao,Yaqu Wang,Xiaoen Xu,et al Mst1 is a interacting protein thatmediates PHLPPs’induced apoptosis.Molecular Cell,2010,38:512-523
（26）Ong,S.E. et al.Stable isotope labeling bu amino acides in cellculture,SILAC,as simple and accutate approach to expression proteomics.2006Mol.Cell.Proteomics 1,376-386
（27）Blagoev,B.et al.A proteomics strategy to elucidate functional protein-proteininteractions applied to EGF signaling.2004 Nature Biotechnol.21.315-318
（28）Mann,M.Functional and quantitative proteomics using SILAC.NatureRev.Mol.Cell Biol.7.952-958(2006)
（29）Everley,P.A. et al. Enhanced analysis of metastatic prostate cance using stableisotopes and high mass accuracy instrumentation.2006 J.ProteomeRes.5.1224-1231
（30）Trinkle-Mulcahy,L. et al.Repo-Man recruits PP1r to chromatin and is essentialfor cell viability ,2006 J.Cell Biol.172,679-692
（31）Kruger,M.et al.SILAC mouse for quantitative proteomics uncovers kindling-3as an essential factor for red blood cell function. Cell 134,353-364(2008)
（32）Geiger,T.,Cox,J.Pawel,O.,Wisniewski,J.R.&Mann,M. Super SILAC mix forquantitative proteomics of human tumor tissue.Nature methods7,383-385(2010)
（33）Ranish,J.A,et al.The study of macromolecular complexes by quantitativeproteomics.2003Nature Genet 33,349-355
（34）Schulze,W.X.,Man,M.A novel proteomic screen for peptide-proteininteractions.2004.J.Biol.Chem.279.10756-10764
（35）Gingras,A.C.,Aebersold,R.&Raught,B. Advances in protein complex analysisusing mass spectrometry.J.physiol.563,11=21(2005)
（36）Ong,S.E, Mann,M., Mass spectrometry-based proteomics turnsquantitative.Nature Chem.boil.1.252-262(2005)
（37）Foster,L.J.et al.Insulin-dependent interactions of proteins with GLUT4reverled through stable isotope labeling by amino acids in cellculture(SILAC).J.Proteome Res.5.64-75(2006)
（38）Blagoev B,Kratchmaroval I,Ong,S.E.,et al. A proteomics strategy to elucidatefunctional protein-protein interactions applied to EGF signaling. NatBiotechnol, 2003,21:315-318
（39）Selbach,M.,Mann,M.,Protein interaction screening by quantitativeimmunoprecipitation combined with knock-down(QUICK).2006 NatureMethods 3,29 Oct 2006
（40）Xiaorong,W.,Lan H.,Identifying Dynamic interactors of protein complexes byquantitative mass spectrometry.Mol.Cellular Proteomics,2008;7(1),46-57
（41）Fan,L.,Wang,X.,Yamoah,K.,et al.Characterization of the human COP9signalosome complex using affinity purification and massspectrometry,J.Proteome Res.&,4914-4925
（42）Mousson F,Kolkman A, Pijnappel W.W.M.P.,et al.Quantitative proteomicsreveals regulation of dynamic components within TATA-binding protein(TBP)transcription complexes.Mol.Cell Proteomics,2008,7:845-852
（43）Seebacher J,Mallick P,Zhang N,Eddes JS,et al.Protein cross-linking analysisusing mass spectrometry,isotope-coded cross-linkers,and integratedcomputational data processing.J Proteome Res 2006,5:2270`-2282
（44）Sinz A.,Chemical cross-linking and mass spectrometry to mapthree-dimensional protein structures and protein-protein interactions.MassSpectrom Rev 2006,25:663-682
（45）Schulze,W.X.,Mann,M.,A novel proteomic screen forpeptide-proteininteractions.J.Biol.Chem.2004,279：10756-10764
（46）Falk,B., Marion,S.,Mario,M.,Mathias M., Unbiased RNA-protein interactionscreen by quantitative proteomics.PNAS,2009,106(26)10626-10631