蛋白质组学

牛启提供的蛋白质组学技术服务主要包括:蛋白质定量分析、蛋白质相对定量分析、蛋白质绝对定量分析 和 蛋白质修饰分析等。

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2016年01月01日

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深圳很多高校和医院,因为时间或实验条件,有些人在提问:蛋白质组学技术服务的公司?深圳哪家公司提供的蛋白质组学技术比较靠谱?蛋白质定量分析服务公司?深圳哪家公司专业做蛋白质?蛋白质相对定量分析服务? 蛋白质绝对定量分析服务公司哪家专业? 蛋白质修饰分析服务哪家公司真实可靠?深圳哪家公司承接整体蛋白质实验 ?


牛启提供的蛋白质组学技术服务主要包括:


一 、蛋白质定量分析


二、蛋白质相对定量分析;


三、蛋白质绝对定量分析;


四、蛋白质修饰分析等



一、蛋白质定量分析;

1.蛋白质全谱分析也可称为质谱shotgun分析,指的是组分分析,研究对象是完整的组织、体液或其提取物,其目的在于鉴定出尽可能多的肽和蛋白质分子。

 

2. 蛋白质胶条/混合液鉴定:利用LC-ESI-MS/MS蛋白鉴定技术对胶条样本(即SDS-PAGE样本)、IP、co-IP、Pull-down等纯化溶液等中等复杂样本进行蛋白鉴定。

3. 蛋白质胶点鉴定:利用MALDI-TOF/TOF蛋白鉴定技术对考/银染的2D或DIGE胶点样本或者较纯的蛋白样本进行蛋白鉴定。


蛋白质定性分析分为两种策略:

(1) top-down分析策略:完整的全长蛋白质经过离子化后进入质量分析器,通过测定蛋白质离子质量和串联质谱分析鉴定蛋白质序列。

(2) bottom-up分析策略:存在于溶液或者凝胶电泳条带中的蛋白质经过蛋白酶(如胰蛋白酶)酶解消化成较小肽段片段,这些肽段再进入质量分析器。最后通过肽指纹图谱或者串联质谱法进行肽段鉴定,该方法是通过对肽段序列的鉴定推断蛋白质序列。


目前,bottom-up分析策略被更广泛的应用于蛋白质定性分析工作。

根据使用仪器可分为:MALDI-TOF/TOF

                                          LC-ESI-MS/MS

根据样本类型可分为:蛋白质全谱分析(即shotgun分析)

                                          蛋白质胶条/混合液分析

                                          蛋白质胶点分析


参考案例


[1]       Zhao B, Wang HB, et al. Transport of receptors, receptor signaling complexes and ion channels via neuropeptide-secretory vesicles. Cell Res. 2011; 21(5): 741- 53.

[2]       Liu J, Zheng S, et al. Protein profiles of the midgut of Spodoptera litura larvae at the sixth instar feeding stage by shotgun ESI-MS approach. J Proteome Res. 2010; 9(5): 2117-47.

[3]       Tamura K, Fukao Y, et al. Identification and characterization of nuclear pore complex components in Arabidopsis thaliana. Plant Cell. 2010; 22(12) 4084-97.

[4]       Wang ZQ, Wang L, et al. Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach. J Proteomics. 2012; 75(8): 2375-83.

[5]       Wang X, Bian Y, et al. A comprehensive differential proteomic study of nitrate deprivation in Arabidopsis reveals complex regulatory networks of plant nitrogen responses. J Proteome Res. 2012; 11(4): 2301-15.

[6]       Michta E, Ding W, et al. Proteomic approach to reveal the regulatory function of aconitase AcnA in oxidative stress response in the antibiotic producer Streptomyces viridochromogenes Tü494. PLoS One. 2014; 9(2).

[7]       Shen J, Abel EL, et al. Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice. Carcinogenesis. 2012; 33(11): 2208-19.



二、蛋白质相对定量分析;


相对定量的目的是测定目的蛋白在两个或多个样本中的表达量的相对比例,而不需要知道它们在每个样本中的表达量。


举例来说,如果研究项目中包括处理过的和未经处理的对照样本,通常可以将未经处理的样本指定为基准,规定其目的蛋白浓度为 100%,将经处理的样本的定量结果除以对照样品的定量结果,就可以计算各个处理样本的蛋白含量相对于未处理样品的百分比。


参考案例:

[1] Yang QS, Wu JH, et al. Quantitative proteomic analysis reveals that antioxidation mechanisms contribute to cold tolerance in plantain (Musa paradisiacal L.; ABB Group)            seedlings. Mol Cell Proteomics. 2012; 11(12): 1853-69.

[2] Leivonen SK, Rokka A, et al. Identification of miR-193b targets in breast cancer cell and systems biological analysis of their functional impact. Mol Cell

[3]   Thompson, A, et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem. 2003; 75(8):1895-1904.

[4]   Dayon, L, et al. Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem. 2008; 80(8):2921-31.

[5]   Zhuang, G, et al. Phosphoproteomic analysis implicates the mTORC2-FoxO1 axis in VEGF signaling and feedback activation of receptor tyrosine kinases. Sci Signal. 2013; 16;  6(271):ra25.

[6]  Niemann M, Wiese S, et al. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology. Mol             Cell  Proteomics. 2013; 12(2): 515-28.

[7] Vanderschuren H, Nyaboga E, et al. Large-Scale Proteomics of the Cassava Storage Root and Identification of a Target Gene to Reduce Postharvest Deterioration. Plant          Cell.  2014.

[8]    Cholewa BD, Pellitteri-Hahn MC, et al. Large-Scale Label-Free Comparative Proteomics Analysis of Polo-Like Kinase 1 Inhibition via the Small-             Molecule Inhibitor BI 6727 (Volasertib) in BRAFV600E Mutant Melanoma Cells. J Proteome Res. 2014 Jun 9.

[9] Kim JY, Welsh EA, et al. Dissection of TBK1 signaling via phosphoproteomics in lung cancer cells. Proc Natl Acad Sci U S A. 2013; 110(30): 12414-9.

[10]  Sobczyk GJ, Wang J, et al. SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale. Nature Commun.         2014; 5: 3319.

[11]  Ravikumar V, Shi L, et al. Quantitative phosphoproteome analysis of Bacillus subtilis reveals novel substrates of the kinase PrkC and phosphatase PrpC. Mol Cell Proteomics.  2014 Jan 5.

[12] Haneda T, Sugimoto M, et al. Comparative proteomic analysis of Salmonella enteric serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required  for intracellular survival in macrophages. BMC Microbiol. 2010; 10: 324.

[13]   Wang X, Bian Y, et al. A comprehensive differential proteomic study of nitrate deprivation in Arabidopsis reveals complex regulatory networks of plant nitrogen responses. J   Proteome Res. 2012; 11(4): 2301-15.

[14]   Gromova I, Gromov P, et al. Proteomic profiling of triple-negative breast carcinomas in combination with a three-tier orthogonal technology approach identifies Mage-        A4 as   potential therapeutic target in estrogen receptor negative breast cancer. Mol Cell Proteomics. 2013; 12(2): 381-94.

[15]    Samuel MA, Tang W, et al. Proteomic analysis of Brassica stigmatic proteins following the self-incompatibility reaction reveals a role for microtubule dynamics during        pollen    responses. Mol Cell Proteomics. 2011; 10(12).

[16]    De la Cuesta F, Alvarez-Llamas, et al. A proteomic focus on the alterations occurring at the human atherosclerotic coronary intima. Mol Cell Proteomics. 2011; 10(4).

[17]    Hong Y, Peng J, et al. Proteomic analysis of Schistosoma japonicum Schistosomulum proteins that are differentially expressed among hosts differing in their          susceptibility to    the infection. Mol Cell Proteomics. 2011; 10(8).



三、蛋白质绝对定量分析;


目前基于质谱的绝对定量蛋白质组学研究主要是指靶向蛋白质组学。靶向蛋白质组学分析,是指对目标蛋白质(或修饰肽段)进行定性和/或定量分析,或者用于验证大规模蛋白质组学的结果。基于质谱的靶向蛋白质组学分析方法,由于没有物种限制并具备多目标同时分析能力等优势,在相关研究领域已逐渐受到越来越多的关注和应用。其技术方法经历了从传统的SRM/MRM(选择性/多反应监视,selected / multiple reaction monitoring)到PRM(平行反应监视,parallel reaction monitoring)的发展历程。


绝对定量蛋白质组学应用方向:

验证定量蛋白质组学结果           验证翻译后修饰蛋白质组学结果


目标蛋白质相对定量分析           目标蛋白质绝对定量分析


诊断标志物靶向筛选              诊断标志物验证与绝对定量


验证基因表达产物



参考案例:


[1] Quantitative Profiling of Protein Tyrosine Kinases in Human Cancer Cell Lines by Multiplexed Parallel Reaction Monitoring Assays.  Mol Cell Proteomics. 2016 15(2):682-91.

PRM同时监测83个酪氨酸激酶表达。

[2] Proteogenomic characterization of human colon and rectal cancer. Nature. 2014; 513(7518): 382-7.

PRM技术验证基因突变表达产物



四、蛋白质修饰分析等


生物体中许多至关重要的生命进程不仅由蛋白质的相关丰度控制,还会被各种时空特意分布、可逆的翻译后修饰调控,因此揭示翻译后修饰的发生规律是解析蛋白质复杂多样的生物功能的一个重要前提。


修饰蛋白质组学技术方法及应用:


参考案例:



[1]   Hutchinson EC, Denham EM, et al. Mapping the phosphoproteome of influenza A and B viruses by mass spectrometry. PLoS Pathog. 2012; 8(11).

[2]   Chumbalkar V, Latha K, et al. Analysis of phosphotyrosine signaling in glioblastoma identifies STAT5 as a novel downstream target of DeltaEGFR. J Proteome Res. 2011;          10(3): 1343-52.

[3]  Wang S, Wang J, et al. PKC-mediated USP phosphorylation at Ser35 modulates 20-hydroxyecdysone signaling in Drosophila. J Proteome Res. 2012; 11(12): 6187-96.

[4]  Nguyen TH, Brechenmacher L, et al. Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum. Mol Cell Proteomics. 2012;        11(11): 1140-55.

[5] Lee DH, Goodarzi AA, et al. Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response. EMBO J. 2012; 31(10): 2403-15.

[6]  Tsigankov P, Gherardini PF, et al. Regulation dynamics of Leishmania differentiation: deconvoluting signals and identifying phosphorylation                 trends. Mol Cell Proteomics. 2014 Apr 16. 

[7] Zielinska DF, Gnad F, et al. Mapping N-glycosylation sites across seven evolutionarily distant species reveals a divergent substrate proteome despite a common core             machinery.    Mol Cell. 2012; 46(4): 542-8.

[8] Guo A, Gu H, et al. Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation. Mol Cell Proteomics. 2014; 13(1): 372-87.

[9] Tong.Z, Kim MS, et al. Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomic in Yeast. Mol Cell Proteomics. 2014; 13(8):      1979-  92.






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