Quantitative Protease Cleavage Site Profiling using Tandem-Mass-Tag Labeling and LC-MALDI-TOF/TOF MS/MS Analysis
Quantitative Protease Cleavage Site Profiling using Tandem-Mass-Tag Labeling and LC-MALDI-TOF/TOF MS/MS Analysis
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DOI：
10.1021/pr201051e
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发表时间：
2012-03-01
影响因子：
4.4
通讯作者：
Tholey, Andreas
中科院分区：
文献类型：
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作者：
Jakoby, Thomas;van den Berg, Bart H. J.;Tholey, Andreas
切割位点特异性和活性的知识是理解蛋白酶功能的主要先决条件。基于最近提出的一种用于蛋白质组衍生肽库中切割位点的蛋白质组学鉴定（PICS）方法，我们开发了一种同量异位素标记的定量LC-MALDI-TOF/TOF MS/MS方法（Q-PICS），用于同时测定切割位点特异性和蛋白水解事件的稳健相对定量。对于GluC-蛋白酶，在酵母蛋白质组衍生的肽库中鉴定了737个切割位点; 94.0%显示出典型的GluC特异性，用于谷氨酰和谷氨酰残基处的肽键。六重串联质量标记策略允许在单次运行中同时进行三次重复，保证了定量测量的高置信度和稳健统计。使用Q-PICS的定量能力，我们进行了比较的GluC在两种不同的缓冲液系统中的切割位点特异性。结果支持早期的研究结果，描述缓冲系统之间的明显差异可能是由碳酸氢盐对整体GluC活性的抑制作用引起的，并且与Asp-X键相比，Glu-X键的偏好与所使用的缓冲系统无关。
Knowledge of cleavage site specificity and activity are major prerequisites for understanding protease function. On the basis of a recently presented approach for proteomic identification of cleavage sites (PICS) in proteome-derived peptide libraries, we developed an isobaric labeling quantitative LC-MALDI-TOF/TOF MS/MS approach (Q-PICS) for simultaneous determination of cleavage site specificity and robust relative quantification of proteolytic events. For GluC-protease, 737 cleavage sites were identified in a yeast proteome-derived peptide library; 94.0% showed the typical GluC specificity for peptide bonds at glutamyl and aspartyl residues. The six-plex tandem mass tagging strategy allowed for three simultaneous replicates in a single run, guaranteeing high confidence and robust statistics for quantitative measurements. Using the quantitative capacity of Q-PICS, we performed a comparison of cleavage site specificity of GluC in two different buffer systems. The results support earlier findings describing that apparent difference between the buffer systems are probably caused by the inhibitory effect of bicarbonate on the overall GluC activity and that the preference for Glu-X bonds compared to Asp-X bonds is independent of the buffer system used.