Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling.
Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling.
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DOI：
10.1021/ac101284c
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发表时间：
2010-07-01
影响因子：
7.4
通讯作者：
Blonder, Josip
中科院分区：
文献类型：
--
作者：
Ye, Xiaoying;Luke, Brian T.;Johann, Donald J., Jr.;Ono, Akira;Prieto, DaRue A.;Chan, King C.;Issaq, Haleem J.;Veenstra, Timothy D.;Blonder, Josip
肽的差异 18O/16O 稳定同位素标记依赖于 H218O 存在下羧基末端酶催化的氧交换，已广泛用于肽/蛋白质的相对定量。胰蛋白酶水解在自下而上鸟枪式蛋白质组学中的作用和低廉的试剂成本，使得胰蛋白酶催化的 18O 消化后交换成为一种方便且经济的稳定同位素标记方法。然而，众所周知，胰蛋白酶催化的羧基末端 18O 交换在许多情况下是不均匀/不完整的。 18O 交换/掺入的程度因肽而异，主要是由于不同的酶-底物亲和力。因此，肽比率的准确计算和解释在分析上是复杂的并且在某些方面是有缺陷的。因此，需要一种能够改进对每个差异标记肽对的实际 18O 掺入测量的计算方法。在这方面，我们开发了一种算法方法，该方法依赖于梯形规则来整合特定肽离子上所有检测到的同位素物种在保留时间内的峰值强度，这使同位素流形符合泊松分布。计算流形拟合的最佳值，然后通过进化编程导出 18O/16O 比率。该算法使用胰蛋白酶催化的 18O 消化后交换进行测试，以先验确定的比例差异标记牛血清白蛋白 (BSA)。利用这种严格的数学方法，准确性和精确度都得到了提高。利用这种算法技术，我们通过考虑不同程度的消化后 18O 交换引起的伪影，证明了该方法准确计算差异标记 BSA 肽的 18O/16O 比率的有效性。我们进一步证明了该方法在对从表达野生型 HIV-1 Gag 及其非肉豆蔻酰化突变体的细胞中分离的耐去污剂膜微结构域 (DRMM) 进行大规模蛋白质组学定量时准确计算 18O/16O 比率的有效性。
Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs, has made trypsin-catalyzed 18O post-digestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin–catalyzed 18O post-digestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both, accuracy and precision are improved utilizing this rigorous mathematical approach. Utilizing this algorithmic technique, we demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios for differentially labeled BSA peptides, by accounting for artifacts caused by a variable degree of post-digestion 18O exchange. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its non myristylated mutant.
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影响因子：
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作者：
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通讯作者：
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