Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.
Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.
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DOI：
10.1074/mcp.m000050-mcp201
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发表时间：
2010-05
期刊：
影响因子：
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通讯作者：
Overall CM
中科院分区：
文献类型：
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作者：
Prudova A;auf dem Keller U;Butler GS;Overall CM
蛋白水解酶是一种主要的蛋白质翻译后修饰，通过改变蛋白质结构影响蛋白质功能，并通过截断蛋白质序列改变蛋白质组学分析的蛋白质的多肽特征。为了在蛋白质组的基础上鉴定这种修饰和缩短的蛋白酶产生的neo-N末端，我们开发了一种用于相对和绝对定量的完整蛋白质等压标签(ITRAQ)标记方法，该方法同时标记和阻断包括蛋白质N-末端和赖氨酸侧链在内的所有伯胺。阻断赖氨酸限制了胰酶对精氨酸的裂解，从而有效地延长了蛋白水解性截短肽的长度，从而改进了MS/MS分析和多肽鉴定。将iTRAQ全蛋白标记与底物末端胺同位素标记(iTRAQ-Tail)相结合，通过阻断成熟的原始N-末端和neo-N-末端的负选择来丰富N-末端具有许多优点。它可以同时表征蛋白质的天然N-末端及其N-末端修饰，以及蛋白质分解产物和裂解位点的鉴定。此外，iTRAQ-Tail还支持多达8个样本的多重N-末端组学分析，并允许在MS2模式下进行定量，从而防止了光谱复杂性的增加，并通过低丰度蛋白质的信号放大扩大了蛋白质组的覆盖范围。我们比较了两种密切相关的基质金属蛋白酶-2(明胶酶A)和基质金属蛋白酶-9(明胶酶B)在成纤维细胞分泌蛋白中的底物降解情况。在与1,054个蛋白质相对应的3,152个独特的N-末端多肽中，我们在相同的条件下检测到201个对基质金属蛋白酶-2的切割产物，而出人意料的是只有19个同源的基质金属蛋白酶-9的切割产物。已鉴定和生化验证的新底物包括胰岛素样生长因子结合蛋白-4、补体C1r组分A、Galectin-1、Dickkopf相关蛋白-3和血栓反应蛋白-2。因此，使用iTRAQ-Tail的N-末端组学分析将明胶酶与血管生成中的新作用机制联系起来，并揭示了这两种非常相似的蛋白酶的底物谱系中的意外限制。
Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.