Bunkenborg Jakob, Matthiesen Rune
Alphalyse, Odense, Denmark.
Computational and Experimental Biology Group, CEDOC, Chronic Diseases Research Centre, NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal.
Methods Mol Biol. 2020;2051:199-230. doi: 10.1007/978-1-4939-9744-2_8.
Tandem mass spectrometry provides a sensitive means of analyzing the amino acid sequence of peptides and modified peptides by providing accurate mass measurements of precursor and fragment ions. Modern mass spectrometry instrumentation is capable of rapidly generating many thousands of tandem mass spectra and protein database search engines have been developed to match the experimental data to peptide candidates. In most studies there is a schism between discarding perfectly valid data and including nonsensical peptide identifications-this is currently managed by establishing a false discovery rate (FDR) but for modified peptides it calls for an understanding of tandem mass spectrometry data. Manual evaluation of the data and perhaps experimental cross-checking of the MS data can save many months of experimental work trying to do biological follow-ups based on erroneous identifications. Especially for posttranslationally modified peptides there is a need for careful consideration of the data because search algorithms seldom have been optimized for the identification of modified peptides and because there are many pitfalls for the unwary. This chapter describes some of the issues that should be considered when interpreting and validating tandem mass spectra and gives some useful tables to aid in this process.
串联质谱法通过提供前体离子和碎片离子的精确质量测量，为分析肽段和修饰肽段的氨基酸序列提供了一种灵敏的方法。现代质谱仪器能够快速生成成千上万的串联质谱图，并且已经开发了蛋白质数据库搜索引擎，以将实验数据与候选肽段进行匹配。在大多数研究中，在丢弃完全有效的数据和纳入无意义的肽段鉴定之间存在分歧——目前通过建立错误发现率（FDR）来管理，但对于修饰肽段，这需要对串联质谱数据有所了解。手动评估数据以及可能对质谱数据进行实验交叉核对，可以节省基于错误鉴定进行生物学后续研究的数月实验工作。特别是对于翻译后修饰的肽段，需要仔细考虑数据，因为搜索算法很少针对修饰肽段的鉴定进行优化，而且对于粗心的人来说存在许多陷阱。本章描述了在解释和验证串联质谱图时应考虑的一些问题，并给出了一些有用的表格以辅助这一过程。
