ASPIC, a novel MS technology, applied to degradomics

描述（由申请人提供）： 这两个阶段的拨款提案旨在开发和完善一种新的质谱技术，称为氨基末端固相同位素编码（ASPIC），用于复杂混合物中多肽的定量和鉴定。我们将专注于ASPIC在生物液体和完整细胞中的蛋白水解事件中的应用，但预计我们的技术以后将在蛋白质组学和细胞生物学中得到更广泛的应用。我们的技术的关键特征是选择性地将两种平行多肽混合物的游离N-末端分别偶联到固体支持物上，所述固体支持物含有化学性质相同但分子量相差6- 9-Da的胺反应性部分。可以洗涤共价偶联的混合物，用胰蛋白酶或其他蛋白酶消化，定量洗脱，并通过二维或三维液相色谱法和串联质谱法（2D或3D LC-MS/MS）解析和分析。两个样品之间蛋白水解的任何差异，即“新”N-末端的产生，将被视为峰高的变化，而“旧”N-末端在两个样品之间将保持相等。由于即使是2D LC-MS/MS也可以很容易地分辨至少162，000个肽，以自动化方式鉴定差异峰高并对其进行测序，因此我们预期获得样品蛋白水解状态（“降解组”）的“快照”。 该项目分为初始基础技术开发阶段（第一阶段，R21）和随后的技术验证和应用阶段（第二阶段，R33）。第一阶段将开发和优化ASPIC化学，并使用血液凝固作为概念验证进行验证。从第一阶段到第二阶段的过渡将取决于确定的里程碑的实现。II期将使用优化的技术来确定构成（i）HEK 293细胞凋亡和（ii）T细胞活化过程中信号通路的蛋白酶和蛋白酶底物。

DESCRIPTION (provided by applicant): This two-phase grant proposal seeks to develop and perfect a new mass spectrometry technique, termed Amino-terminal Solid-Phase Isotope Coding (ASPIC), for quantitation and identification of polypeptides in complex mixtures. We will focus on the application of ASPIC to proteolytic events in biological fluids and in intact cells, but anticipate that our technology will later find much broader use in proteomics and cell biology. The key feature of our technology is to selectively couple free N-termini of two parallel polypeptide mixtures individually to a solid support that contains amine-reactive moieties of identical chemical nature, but differing by 6- to 9-Da in molecular mass. The covalently coupled mixture can be washed, digested with trypsin or other proteases, quantitatively eluted, and resolved and analyzed by 2- or 3-dimensional liquid chromatography and tandem mass spectrometry (2D or 3D LC-MS/MS). Any differences in proteolysis, i.e. creation of 'new' N-termini, between two samples will be seen as changes in peak heights, while 'old' N-termini will remain equal between the two samples. Since even 2D LC-MS/MS can readily resolve at least 162,000 peptides, identify differential peak heights in an automated fashion and sequence them, we anticipate obtaining a 'snapshot' of the proteolytic state ('degradome') of the sample. This project is divided into an initial basic technology development phase (Phase I, R21) and a subsequent technology validation and application phase (Phase II, R33). Phase I will develop and optimize the ASPIC chemistry and validate it using blood coagulation as a proof of concept. The transition from Phase I to Phase II will depend on the fulfillment of defined milestones. Phase II will use the optimized technology to determine the proteases and protease substrates that constitute the signaling pathways during (i) apoptosis of HEK293 cells and (ii) T-cell activation.