Burns Kyle M, Sarpe Vladimir, Wagenbach Mike, Wordeman Linda, Schriemer David C
Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington, 98195-7290.
Protein Sci. 2015 Aug;24(8):1313-24. doi: 10.1002/pro.2707. Epub 2015 May 29.
Water-mediated hydrogen exchange (HX) processes involving the protein main chain are sensitive to structural dynamics and molecular interactions. Measuring deuterium uptake in amide bonds provides information on conformational states, structural transitions and binding events. Increasingly, deuterium levels are measured by mass spectrometry (MS) from proteolytically generated peptide fragments of large molecular systems. However, this bottom-up method has limited spectral capacity and requires a burdensome manual validation exercise, both of which restrict analysis of protein systems to generally less than 150 kDa. In this study, we present a bottom-up HX-MS(2) method that improves peptide identification rates, localizes high-quality HX data and simplifies validation. The method combines a new peptide scoring algorithm (WUF, weighted unique fragment) with data-independent acquisition of peptide fragmentation data. Scoring incorporates the validation process and emphasizes identification accuracy. The HX-MS(2) method is illustrated using data from a conformational analysis of microtubules treated with dimeric kinesin MCAK. When compared to a conventional Mascot-driven HX-MS method, HX-MS(2) produces two-fold higher α/β-tubulin sequence depth at a peptide utilization rate of 74%. A Mascot approach delivers a utilization rate of 44%. The WUF score can be constrained by false utilization rate (FUR) calculations to return utilization values exceeding 90% without serious data loss, indicating that automated validation should be possible. The HX-MS(2) data confirm that N-terminal MCAK domains anchor kinesin force generation in kinesin-mediated depolymerization, while the C-terminal tails regulate MCAK-tubulin interactions.
涉及蛋白质主链的水介导氢交换（HX）过程对结构动力学和分子相互作用敏感。测量酰胺键中的氘摄取可提供有关构象状态、结构转变和结合事件的信息。越来越多地，通过质谱（MS）从大分子系统的蛋白水解产生的肽片段中测量氘水平。然而，这种自下而上的方法具有有限的光谱容量，并且需要繁重的手动验证操作，这两者都将蛋白质系统的分析限制在通常小于150 kDa的范围内。在本研究中，我们提出了一种自下而上的HX-MS(2)方法，该方法提高了肽鉴定率，定位了高质量的HX数据并简化了验证。该方法将一种新的肽评分算法（WUF，加权独特片段）与肽片段化数据的独立数据采集相结合。评分纳入了验证过程并强调鉴定准确性。使用来自用二聚体驱动蛋白MCAK处理的微管构象分析的数据说明了HX-MS(2)方法。与传统的由Mascot驱动的HX-MS方法相比，HX-MS(2)在肽利用率为74%时产生的α/β-微管蛋白序列深度高出两倍。Mascot方法的利用率为44%。WUF评分可以通过错误利用率（FUR）计算来约束，以返回超过90%的利用率而不会有严重的数据丢失，这表明自动验证应该是可行的。HX-MS(2)数据证实，N端MCAK结构域在驱动蛋白介导的解聚中锚定驱动蛋白的力产生，而C端尾巴调节MCAK-微管蛋白相互作用。
