Patteson K G, Rodicio L P, Limbach P A
232 Choppin Hall, Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
Nucleic Acids Res. 2001 May 15;29(10):E49-9. doi: 10.1093/nar/29.10.e49.
A new method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the direct analysis of the mass-silent post-transcriptionally modified nucleoside pseudouridine in nucleic acids has been developed. This method utilizes 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to derivatize pseudouridine residues. After chemical derivatization all pseudouridine residues will contain a 252 Da 'mass tag' that allows the presence of pseudouridine to be identified using mass spectrometry. Pseudouridine residues can be identified in intact nucleic acids by obtaining a mass spectrum of the nucleic acid before and after derivatization. The mass difference (in units of 252 Da) will denote the number of pseudouridine residues present. To determine the sequence location of pseudouridine, a combination of enzymatic hydrolysis and mass spectrometric steps are used. Here, MALDI analysis of RNase T1 digestion products before and after modification are used to narrow the sequence location of pseudouridine to specific T1 fragments in the gene sequence. Further mass spectrometric monitoring of exonuclease digestion products from isolated T1 fragments is then used for exact sequence placement. This approach to pseudouridine identification is demonstrated using Escherichia coli tRNAS: This new method allows for the direct determination of pseudouridine in nucleic acids, can be used to identify modified pseudouridine residues and can be used with general modification mapping approaches to completely characterize the post-transcriptional modifications present in RNAs.
已开发出一种利用基质辅助激光解吸/电离（MALDI）质谱直接分析核酸中无质量变化的转录后修饰核苷假尿苷的新方法。该方法利用1-环己基-3-（2-吗啉代乙基）碳二亚胺对假尿苷残基进行衍生化。化学衍生化后，所有假尿苷残基将含有一个252 Da的“质量标签”，可通过质谱法鉴定假尿苷的存在。通过获取衍生化前后核酸的质谱图，可以在完整核酸中鉴定假尿苷残基。质量差（以252 Da为单位）将表示存在的假尿苷残基数量。为了确定假尿苷的序列位置，采用了酶促水解和质谱步骤相结合的方法。在此，通过对修饰前后的核糖核酸酶T1消化产物进行MALDI分析，将假尿苷的序列位置缩小到基因序列中的特定T1片段。然后，对分离出的T1片段的核酸外切酶消化产物进行进一步的质谱监测，以进行精确的序列定位。使用大肠杆菌tRNAS证明了这种鉴定假尿苷的方法：这种新方法可以直接测定核酸中的假尿苷，可用于鉴定修饰的假尿苷残基，并可与一般的修饰图谱方法一起使用，以全面表征RNA中存在的转录后修饰。
