Wenner Brett R, Lynn Bert C
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0286, USA.
J Am Soc Mass Spectrom. 2004 Feb;15(2):150-7. doi: 10.1016/j.jasms.2003.10.006.
Quadrupole ion trap scanning parameters for performing bottom-up proteomics in a data-dependent fashion were evaluated on a Finnigan LCQ Deca mass spectrometer. Evaluation of parameters such as the number of averaged full scans, the number of averaged MS/MS scans, and ion injection times were necessary for acquiring high quality MS/MS spectra that yield favorable b and y ion coverage and high correlation to proteins using database searching algorithms. In this study, we demonstrated how the duty cycle of the mass spectrometer affects the number of peptides that can be successfully identified by SEQUEST using a model system of tryptic BSA peptides to mimic a typical complex mixture associated with bottom-up proteomics. The number of averaged scans and the duration of ion accumulation in the trap had a significant effect on the quality of acquired MS/MS spectra. For example, by increasing the ion injection time from 500 ms to 600 ms, peptide HLVDEPQNLIK improved from being improperly identified to being correctly identified with a SEQUEST cross-correlation score of 3.60. As a result of these experiments, we have devised the following set of ion trap parameters for performing bottom-up proteomics analysis in our laboratory: Three averaged full scans, five averaged MS/MS scans, and a maximum ion injection time of 600 ms.
在Finnigan LCQ Deca质谱仪上评估了以数据依赖方式进行自下而上蛋白质组学分析时的四极杆离子阱扫描参数。为了获得高质量的串联质谱（MS/MS）谱图，从而在使用数据库搜索算法时能产生良好的b离子和y离子覆盖率并与蛋白质具有高度相关性，有必要对诸如平均全扫描次数、平均MS/MS扫描次数和离子注入时间等参数进行评估。在本研究中，我们使用胰蛋白酶消化的牛血清白蛋白（BSA）肽的模型系统来模拟与自下而上蛋白质组学相关的典型复杂混合物，展示了质谱仪的占空比如何影响通过SEQUEST成功鉴定的肽段数量。平均扫描次数和阱中离子积累的持续时间对所采集的MS/MS谱图质量有显著影响。例如，将离子注入时间从500毫秒增加到600毫秒后，肽段HLVDEPQNLIK从鉴定错误转变为正确鉴定，其SEQUEST交叉相关分数为3.60。基于这些实验结果，我们在实验室中设计了以下一组用于进行自下而上蛋白质组学分析的离子阱参数：三次平均全扫描、五次平均MS/MS扫描以及最大离子注入时间600毫秒。
