Automated extraction of backbone deuteration levels from amide H/2 H mass spectrometry experiments
Automated extraction of backbone deuteration levels from amide H/2 H mass spectrometry experiments
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DOI：
10.1110/ps.051774906
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发表时间：
2006-03-01
期刊：
影响因子：
8
通讯作者：
Ten Eyck, LF
中科院分区：
文献类型：
--
作者：
Hotchko, M;Anand, GS;Ten Eyck, LF
已经开发了傅立叶去卷积方法，以明确地确定通过氢/氘（H/D）交换与高分辨率质谱法结合到蛋白质区域或片段中的骨架酰胺氘的量。测定和分析的水平和数量的骨干酰胺交换溶液中提供更多的信息，溶剂的蛋白质的可及性比以前的质心方法，只计算平均氘交换。交换后，蛋白质被消化成肽，作为确定蛋白质局部区域内交换的一种方式。肽在氘化后的质量是天然同位素丰度、快速交换侧链氢（存在于MALDI-TOF H/H-2数据中）和骨架酰胺交换的总和。由于天然同位素丰度和快速交换侧链而去除同位素分布的组分允许精确定量骨架酰胺交换的水平，如来自蛋白激酶A的实例所示。去卷积的结果受到重叠的肽或不一致的质量信封，这些情况下的评估程序进行了讨论。最后，提出了一种确定反交换修正总体的方法，并讨论了在不同情况下它对数据的影响。
A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/H-2 data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.