Optimized Orbitrap HCD for Quantitative Analysis of Phosphopeptides
Optimized Orbitrap HCD for Quantitative Analysis of Phosphopeptides
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DOI：
10.1016/j.jasms.2009.03.019
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发表时间：
2009-08-01
影响因子：
3.2
通讯作者：
Marto, Jarrod A.
中科院分区：
文献类型：
--
作者：
Zhang, Yi;Ficarro, Scott B.;Marto, Jarrod A.
尽管射频四极离子阱在自下而上的蛋白质组学研究中取得了巨大的商业成功，但越来越多的证据表明，用不稳定的翻译后修饰修饰的肽不太适合低能量、共振激发 MS/MS 分析。此外，设计用于基于 MS/MS 的肽定量的多重稳定同位素试剂依赖于对所有前体的低质量片段的准确和稳健的检测。总的来说，这些观察结果表明，束型或串联空间 MS/MS 测量（例如可用的石油传统三重四极杆质谱仪）可以为定量蛋白质组学分析提供有益的品质因数。最近在所有 Orbitrap 质量分析仪附近引入了多极碰撞池，可提供更高能量的碰撞激活解离 (HCD)，并在较宽的质量范围内有效捕获碎片离子。在这里，我们描述了各种仪器和采集后参数的优化，这些参数共同提供了从复杂细胞裂解物中分离的 iTRAQ 标记的磷酸化肽的定量。浓度动态范围为 100:1 的肽很容易定量。我们的结果表明，将碰撞能量适当参数化为前体 m/z 和 z 的函数，可在 iTRAQ 的肽鉴定和相对定量方面提供最佳性能。使用这种方法，我们可以轻松地在人类髓系白血病模型系统中识别 Flt-3 激酶致癌突变体下游的激活信号通路。 (J Am Soc Mass Spectrom 2009, 20, 1425-1434) (C) 2009 美国质谱学会
Despite the tremendous commercial success of radio frequency quadrupole ion traps for bottom-up proteomics studies, there is growing evidence that peptides decorated with labile post-translational modifications are less amenable to low-energy, resonate excitation MS/MS analysis. Moreover, multiplexed stable isotope reagents designed for MS/MS-based quantification of peptides rely on accurate and robust detection of low-mass fragments for all precursors. Collectively these observations Suggest that beam-type or tandem in-space MS/MS measurements, such as that available oil traditional triple quadrupole mass spectrometers, may provide beneficial figures of merit for quantitative proteomics analyses. The recent introduction of a multipole collision cell adjacent to all Orbitrap mass analyzer provides for higher energy collisionally activated dissociation (HCD) with efficient capture of fragment ions over a wide mass range. Here we describe optimization of various instrument and post-acquisition parameters that collectively provide for quantification of iTRAQ-labeled phosphorylated peptides isolated from complex cell lysates. Peptides spanning a concentration dynamic range of 100:1 are readily quantified. Our results indicate that appropriate parameterization of collision energy as a function of precursor m/z and z provides for optimal performance in terms of peptide identification and relative quantification by iTRAQ. Using this approach, we readily identify activated signaling pathways downstream of oncogenic Mutants of Flt-3 kinase in a model system of human myeloid leukemia. (J Am Soc Mass Spectrom 2009, 20, 1425-1434) (C) 2009 American Society for Mass Spectrometry