Resolving the composition of protein complexes using a MALDI LTQ Orbitrap.
Resolving the composition of protein complexes using a MALDI LTQ Orbitrap.
复制标题
DOI：
10.1016/j.jasms.2009.08.026
复制
发表时间：
2010-01
影响因子：
3.2
通讯作者：
Cristea IM
中科院分区：
文献类型：
--
作者：
Luo Y;Li T;Yu F;Kramer T;Cristea IM
目前的生物学研究已经推进了稳健，准确和灵敏的质谱技术的不断发展。MALDI LTQ轨道阱是轨道阱配置的新成员，以其高分辨率和精度而闻名。这种配置提供了MALDI源固有的功能，例如降低光谱复杂性，对污染物的宽容，以及针对目标或假设驱动问题的后续分析的样品保留。在这里，我们研究其性能的特点分离的蛋白质复合物的组成。为了便于评估，我们选择了两个充分表征的复合物从酵母菌Apl 1和NUP 84。手动和自动MS和MS/MS分析很容易解决他们的组合物，与我们以前的报告相比，使用MALDI QqTOF和MALDI IT的蛋白质鉴定的信心增加。CID片段的单电荷肽提供了足够的信息，为确定性的鉴定分离的蛋白质。然后，我们评估了该仪器在分析分离的蛋白质组装体的背景下提供的分辨率、准确度和灵敏度。我们对m/z 4000以下的单电荷离子复杂混合物的分析表明：1）分辨能力与m/z的平方根成反比，动态范围超过4个数量级; 2）内标导致准确度提高，平均绝对质量误差为0.5ppm，分布中心为0 ppm;和3）使用CHCA和DHB基质两者实现亚飞摩尔灵敏度。此外，我们的分析的合成磷酸化肽的混合物显示subfemtomol水平的检测使用中性丢失扫描。
The current biological studies have been advanced by the continuous development of robust, accurate and sensitive mass spectrometric technologies. The MALDI LTQ Orbitrap is a new addition to the orbitrap configurations, known for their high resolving power and accuracy. This configuration provides features inherent to the MALDI source, such as reduced spectra complexity, forgiveness to contaminants, and sample retention for follow-up analyses with targeted or hypothesis-driven questions. Here we investigate its performance for characterizing the composition of isolated protein complexes. To facilitate the assessment, we selected two well characterized complexes from Saccharomyces cerevisiae—Apl1 and Nup84. Manual and automatic MS and MS/MS analyses readily resolved their compositions, with increased confidence of protein identification when compared to our previous reports using MALDI QqTOF and MALDI IT. CID fragmentation of singly-charged peptides provided sufficient information for conclusive identification of the isolated proteins. We then assessed the resolution, accuracy and sensitivity provided by this instrument in the context of analyzing the isolated protein assemblies. Our analysis of complex mixtures of singly-charged ions up to m/z 4000 showed that 1) the resolving power, inversely proportional to the square root of m/z, had over 4 orders of magnitude dynamic range; 2) internal calibration led to improved accuracy, with an average absolute mass error of 0.5 ppm and a distribution centered at 0 ppm; and 3) subfemtomol sensitivity was achieved using both CHCA and DHB matrices. Additionally, our analyses of a synthetic phosphorylated peptide in mixtures showed subfemtomol level of detection using neutral loss scanning.
登录
查看更多内容
影响因子：
4.8
作者：
Cristea, Ileana M.;Carroll, John-William N.;MacDonald, Margaret R.
通讯作者：
MacDonald, Margaret R.
影响因子：
7
作者：
Deng, Changhui;Xiong, Xinghua;Krutchinsky, Andrew N.
通讯作者：
Krutchinsky, Andrew N.
影响因子：
7.4
作者：
Edelson-Averbukh, Marina;Shevchenko, Andrej;Lehmann, Wolf D.
通讯作者：
Lehmann, Wolf D.
影响因子：
7.4
作者：
Bahr, Ute;Ayguen, Hueseyin;Karas, Michael
通讯作者：
Karas, Michael
DOI：
10.1016/s1044-0305(00)00114-8
发表时间：
2000-06-01
影响因子：
3.2
作者：
Krutchinsky, AN;Zhang, WZ;Chait, BT
通讯作者：
Chait, BT