Klingler Diana, Hardt Markus
Boston Biomedical Research Institute, MA, USA.
J Vis Exp. 2013 Feb 20(72):e3891. doi: 10.3791/3891.
Stable isotopes are essential tools in biological mass spectrometry. Historically, (18)O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes(1-3). With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of (18)O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao(4), Miyagi and Rao(5) and Ye et al.(6)). (18)O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques(7). Depending on the protease utilized, (18)O-labeling can result in the incorporation of up to two (18)O-atoms in the C-terminal carboxyl group of the cleavage product(3). The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction(8). In our PALeO (protease-assisted labeling employing (18)O-enriched water) adaptation of enzymatic (18)O-labeling, we utilized 50% (18)O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases(9) and monitor proteolytic reactions(10-11). Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second (18)O-atom. Such "double-labeling" enzymes can be used for postdigestion (18)O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing (18)O-enriched water beyond enzymes and uses acidic pH conditions to introduce (18)O-stable isotope signatures into peptides.
稳定同位素是生物质谱分析中的重要工具。从历史上看，（18）O稳定同位素已被广泛用于研究蛋白水解酶的催化机制（1 - 3）。随着基于质谱的蛋白质组学的出现，利用稳定同位素富集水中的（18）O原子进行酶催化掺入已成为定量比较蛋白质表达水平的常用方法（Fenselau和Yao（4）、Miyagi和Rao（5）以及Ye等人（6）对此进行了综述）。（18）O标记是化学（如iTRAQ、ICAT）和代谢（如SILAC）标记技术的一种简单且低成本的替代方法（7）。根据所使用的蛋白酶不同，（18）O标记可使裂解产物的C末端羧基中掺入多达两个（18）O原子（3）。标记反应可细分为两个独立过程，即肽键裂解和羧基氧交换反应（8）。在我们对酶促（18）O标记的PALeO（使用（18）O富集水的蛋白酶辅助标记）改进方法中，我们使用50%的（18）O富集水以产生独特的同位素特征。结合高分辨率基质辅助激光解吸电离飞行时间串联质谱（MALDI - TOF/TOF MS/MS），这些特征性的同位素包络可用于以高特异性鉴定裂解产物。我们之前已使用PALeO方法检测和表征内源性蛋白酶（9）并监测蛋白水解反应（10 - 11）。由于PALeO编码了蛋白水解裂解反应的核心本质，实验设置简单，且可避免裂解产物的生化富集步骤。PALeO方法可轻松扩展到（i）监测蛋白水解裂解反应动力学的时间进程实验，以及（ii）分析代表生理条件的复杂生物样品中的蛋白水解作用。PALeO - 时间进程实验有助于识别复杂蛋白水解途径反应中的限速加工步骤和反应中间体。此外，PALeO反应使我们能够鉴定诸如丝氨酸蛋白酶胰蛋白酶之类的蛋白水解酶，该酶能够重新结合其裂解产物并催化第二个（18）O原子的掺入。这种“双标记”酶可用于消化后（18）O标记，其中肽仅通过羧基氧交换反应进行标记。我们的第三种策略将使用（18）O富集水的标记扩展到酶以外的情况，并利用酸性pH条件将（18）O稳定同位素特征引入肽中。
