Quantitative Protease Cleavage Site Profiling using Tandem-Mass-Tag Labeling and LC–MALDI-TOF/TOF MS/MS Analysis

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.

掌握切割位点特异性与酶活特性，是解析蛋白酶功能的核心先决条件。基于近期提出的蛋白质组来源肽库切割位点鉴定（proteomic identification of cleavage sites, PICS）技术，我们开发了一种同重同位素标记定量液相色谱-基质辅助激光解吸电离飞行时间串联质谱（LC-MALDI-TOF/TOF MS/MS）方法，即定量切割位点鉴定技术（quantitative PICS, Q-PICS），可同时实现切割位点特异性的测定与蛋白水解事件的精准相对定量。针对GluC蛋白酶，我们在酵母蛋白质组来源的肽库中鉴定出737个切割位点；其中94.0%符合GluC典型的酶切特异性，即优先切割谷氨酸残基与天冬氨酸残基介导的肽键。该六重串联质量标签（tandem mass tagging, TMT）策略可在单次实验中同时完成三次生物学重复，为定量检测提供了高置信度与可靠的统计学支撑。依托Q-PICS的定量能力，我们对比了两种不同缓冲体系中GluC的切割位点特异性。研究结果佐证了此前的相关结论：两种缓冲体系间的表观差异，大概率源于碳酸氢根对GluC整体酶活的抑制作用；而GluC对Glu-X肽键相较于Asp-X肽键的偏好性，与所使用的缓冲体系无关。