Interactome data and databases: different types of protein interaction 周新荣 10708046

outline Introduction Types Methods Databases

What do we mean by protein interaction ? Definition：involve the interaction produced by physical contact between the surfaces of two proteins. Protein relations：multiprotein complexes, common cellular compartments, same signaling pathway, same metabolic pathway, co-expression, genetic co-regulation, or even molecular co-evolution.

Different types of protein interaction Co-interacting proteins, defined as physical interaction : (a) Permanent interaction: proteins forming a stable protein complex that carries out a biomolecular role. (b) Transient interaction: proteins that come together in certain cellular states to undertake a biomolecular function.

Different types of protein interaction 2. Correlated proteins, defined as proteins that are involved in the same biomolecular activity but that do not interact physically: (a) Metabolic correlation: proteins involved in the same metabolic pathway. (b) Genetic correlation: proteins that are encoded by co-expressed or co-regulated genes.

Different types of protein interaction 3. Co-located proteins, defined as proteins that work in the same cellular compartment : (a)Soluble location: proteins placed in the same cellular soluble space. (b) Membrane location: proteins placed in the same cellular membrane.

Methods 1.Experimental approaches to protein-protein interaction networks： （a）two-hybrid system （b） serial analysis of gene expression （c）protein microarrays （d） the systematic isolation of multiprotein complexes previously tagged

Methods 2.Computational approaches to protein–protein interaction networks (b) Proteome level: comparison of orthologous proteins in phylogenetic profiles; identification of proteins with similar phylogenetic trees; identification of correlated mutations between the multiple sequence alignments of pairs of proteins. (a) Genome level: conservation of gene order and neighborhood; identification of domain fusion events for orthologous genes.

Databases of Protein Interactions

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以yeast two-hybrid system为例 系 统 构 建：分别构建含GAL4 BD 和AD 的两个酵母融合蛋白表达载体；建立含特殊基因型、适用于双杂交体分析的酵母菌株

SAGE技术的理论基础 首先，一段来自于任一转录本特定区域的“标签” (Tag)，即长度仅10～14bp的短核苷酸序列，就已包含了足够的信息以特异性地确定该转录本。 例如：一个10碱基的序列能有410=1048576种不同的排列组合，而人类基因组据估计仅编码80000种转录本，因此在理论上每一个10碱基标签就能够代表一种转录本的特征序列。 SAGE Tag CATG AAAAAAAAAAAAAAAAA TTTTTTTTTTTTTTTTT Average 256 base pairs

SAGE技术的理论基础 将短片段两两随机配对，形成双标签体 (Ditag)，在标签的种类、数量极大丰富的情况下，完全相同的双标签体出现的概率趋近于零。 将这些双标签体相互连接、集中形成长的DNA分子，对其进行测序将能得到大量连续的单个标签，并能以连续的数据形式进行处理。这样就可对数以千计的mRNA转录本进行批量分析。而在数据处理时将重复出现的双标签体剔除，即可避免由于PCR反应的非均一化扩增而引入的数据偏差；同时，各转录本的表达水平也就可以用特定标签被测得的次数来进行精确定量了。 SAGE Tag 1 SAGE Tag 3 SAGE Tag 5 CATG CATG CATG SAGE Tag 2 SAGE Tag 4 SAGE Tag 6

protein microarrays 基本原理：将高度密集排列的蛋白分子作为探针点阵固定在固相支持物上，当与待测蛋白样品反应时，可捕获样品中的靶蛋白，再经检测系统对靶蛋白进行定性和定量分析。

串联亲和纯化耦联质谱技术 基本原理：在靶蛋白一端或中部嵌入蛋白质标记（TAP Tag），经过特异性的两步亲和纯化，在生理条件下与靶蛋白相互作用的蛋白质便可洗脱下来，然后用质谱技术对得到的蛋白质复合体进行鉴定。

主要流程： 双重分子标签构建到靶蛋白 ↓ 表达融合蛋白 制备细胞裂解液 IgG柱纯化 TEV蛋白酶的洗脱液洗脱蛋白质复合体 耦联钙调素的亲和柱纯化 洗脱 含EGTA的洗脱液洗脱 质谱鉴定结合蛋白质 主要流程：