2-D电泳与生物质谱技术 组员：游明亮 方国 李健

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1 2-D电泳与生物质谱技术 组员：游明亮 方国 李健

2 利用蛋白质的等电点和分子量通过双向凝胶电泳的方法将各种蛋白质区分开来是一种很有效的手段。它在蛋白质组分离技术中起到了关键作用。
蛋白质组学（proteome）的研究技术 利用蛋白质的等电点和分子量通过双向凝胶电泳的方法将各种蛋白质区分开来是一种很有效的手段。它在蛋白质组分离技术中起到了关键作用。 2D电泳技术 通过测定蛋白质的质量来判别蛋白质的种类。 质谱技术

3 蛋白质组研究的核心技术 双向凝胶电泳－质谱技术 即通过双向凝胶电泳将蛋白质分离 然后利用质谱对蛋白质逐一进行鉴定

4 双向电泳的分类 非变性2D-PAGE 非变性/SDS-2D-PAGE 非变性/还原/SDS-2D-PAGE 变性2D-PAGE

5 二维聚丙烯酰胺凝胶电泳原理介绍 二维聚丙烯酰胺凝胶电泳技术结合了等电聚焦技术（根据蛋白质等电点进行分离）以及SDS－聚丙烯酰胺凝胶电泳技术（根据蛋白质的大小进行分离）.这两项技术结合形成的二维电泳是分离分析蛋白质最有效的一种电泳手段。

6 （Detection/Staining）
双向电泳的操作步骤 样品制备 （Sample preparation） 第一向等电聚焦 （IEF） 第二向SDS 电泳 （SDS-PAGE） 2-DE胶蛋白质点的检测 （Detection/Staining）

7 等电聚焦示意图

8 EttanTM IPGphor 3 等电聚焦电泳系统

9 凝胶成像分析系统 用于对各种凝胶和放射自显影胶片、印记杂交片的分析

10 质谱分析仪 质谱仪(mass spectrometry, MS)是以热电子撞击气体分子，使产生碎片及离子，再经磁场分离，依据质荷之比测量，来决定分子质量的技术。

11 质谱分析仪原理： 通过电离源将蛋白质分子转化为气相。
然后利用质谱分析仪的电场、磁场将具有特定质量与电荷比值(Ｍ/Ｚ值)的蛋白质离子分离开来。 经过离子检测器收集分离的离子，确定离子的Ｍ/Ｚ值，分析鉴定未知蛋白质。

12 纵坐标表示相对强度 横坐标的峰高表示M/Z的离子数目

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14 Introduction： Congenital disorders of glycosylation(CDG) are inherited diseases that can affect not only the N-glycan but also the O-glycan biosynthesis pathway. 2-DE and immunoblotting were applied to the separation and simultaneous detection of the isoforms of the O-glycosylated protein apolipoprotein C-III (apoC-III) and of four N-glycosylated proteins,namely alpha-antitrypsin (AAT),alpha-1 acid glycoprotein (AGP),haptoglobin (Hpt) and transferrin (Trf).

15 Procedure: IEF was carried out using 180mm linear pH3–10 IPG strips and Protean ® IEF Cell. After IEF,the strips were incubated for 10min ine quilibration buffer, before being applied polyacrylamide SDS-PAGE gel for the second protein separation. After2-DE,proteins were blotted onto an NC membrane. Glycoproteins of interest were simultaneously detected with ECL reagents using a mixture of up to five primary rabbit antibodies (anti-apoC-III,anti-AAT,anti-AGP,anti-Hpt,anti-Trf) and anti-rabbit IgG linked to horseradish peroxidase as the secondary antibody. Exposed films and scaned them.

16 Results: Screening congenital disorders of O-glycosylation
FigA Typical2-DE pattern of apoC-III isoforms. FigB Control: the corresponding 2-DE pattern obtained after 1 h of neuraminidase treatment. Spot 1 and 2 disappeared and Spot 0b increased,indicating that Spot 0b is the disorder isoform of Spot 1 and 2.

17 Screening congenital disorders of N-glycosylation
FigA Typical 2-DE patterns obtained after simultaneous detection of apoC-III,AGP,AAT,Hpt,Trf. FigB Control:Enlarged Areas showing marked N-glycan abnormalities in from a patient with CDG-Ia.

18 Conclusion: 2-DE coupled to immunoblotting using a mixture of specific antibodies could be easily and reliably employed for the combined screening of both N- and O-glycosylation disorders in humans.

19 Thank you for your attention!