ASPIC, a novel MS technology, applied to degradomics

描述(由申请人提供)： 这项分两个阶段的拨款计划旨在开发和完善一种新的质谱学技术，称为氨基末端固相同位素编码(ASPIC)，用于对复杂混合物中的多肽进行定量和鉴定。我们将专注于将ASPIC应用于生物液和完整细胞中的蛋白质分解事件，但预计我们的技术稍后将在蛋白质组学和细胞生物学中得到更广泛的应用。我们技术的关键特征是将两个平行多肽混合物的游离N末端分别选择性地偶联到一个固体载体上，该固体载体包含化学性质相同但分子质量不同的6-到9-Da的胺反应部分。共价偶联的混合物可以洗涤，用胰酶或其他酶消化，定量洗脱，并用二维或三维液相色谱和串联质谱仪(2D或3D LC-MS/MS)进行拆分和分析。两个样品之间蛋白质分解作用的任何差异，即新的N-末端的产生，都将被视为峰值高度的变化，而两个样品之间的旧的N-末端将保持相同。由于即使是2D LC-MS/MS也可以很容易地拆分至少162,000个多肽，以自动方式识别不同的峰高并对它们进行排序，因此我们预计可以获得样品的蛋白质分解状态(降解穹顶)的“快照”。 该项目分为初步基础技术开发阶段(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肉冻化学，并使用血液凝固作为概念验证来验证它。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情况。第二阶段将使用优化的技术来确定在(I)HEK293细胞的凋亡和(Ii)T细胞激活过程中构成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.