Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
Nat Biotechnol. 2010 Mar;28(3):281-8. doi: 10.1038/nbt.1611. Epub 2010 Mar 7.
Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.
有效的蛋白质组学策略，可以将蛋白质的 N 末端与蛋白酶切割产物的 N 末端区分开来，这将加速鉴定具有广泛或未知特异性的蛋白酶的底物。我们的方法命名为末端胺同位素标记的底物（TAILS），通过使用树枝状多甘油醛聚合物来去除胰蛋白酶和 C 末端肽，从而解决了这一挑战。我们通过串联质谱分析未结合的天然乙酰化、环化或标记的蛋白质及其蛋白酶切割产物的 N 末端，并使用肽同位素定量来区分感兴趣的蛋白酶的底物和背景蛋白水解的产物。我们在小鼠成纤维细胞的分泌产物中鉴定了 731 个乙酰化和 132 个环化的 N 末端，以及 288 个基质金属蛋白酶（MMP）-2 切割位点。我们进一步通过分析小鼠炎症性支气管肺泡液证明了我们的策略在复杂样品中连接具有明确生物学途径的蛋白酶的潜力，并表明在 MCF-7 人乳腺癌细胞中表达定义不明确的乳腺癌蛋白酶 MMP-11 可切割内质网蛋白和免疫调节剂及凋亡诱导剂半乳糖凝集素-1。
