Bentzley C M, Johnston M V, Larsen B S
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Anal Biochem. 1998 Apr 10;258(1):31-7. doi: 10.1006/abio.1998.2569.
Calf spleen phosphodiesterase cleaves oligonucleotide strands in a stepwise manner from the 5' end and can be used in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry to perform ladder sequencing. The relative intensities of ladder peaks in the mass spectra of a series of 5-mers and 7-mers show that the rate of digestion is influenced by strand sequence. Sequences terminating in A or G at the 5' end are found to react two to three times faster than sequences terminating in C or T. The reactivity of the terminal base is also influenced by the sequence beyond the 5' end. When the third base from the 5' end is A or G, removal of the first and second bases is faster than when the third base is C or T. A method is described which permits reaction rates to be quantitatively determined from the time dependences of ladder peaks in the MALDI spectra. A similar approach could be used for mechanistic studies.
小牛脾磷酸二酯酶从5'端逐步切割寡核苷酸链，可与基质辅助激光解吸电离（MALDI）质谱联用进行阶梯测序。一系列5聚体和7聚体质谱中阶梯峰的相对强度表明，消化速率受链序列影响。发现5'端以A或G结尾的序列反应速度比以C或T结尾的序列快两到三倍。末端碱基的反应性也受5'端以外序列的影响。当5'端第三个碱基为A或G时，第一个和第二个碱基的去除速度比第三个碱基为C或T时更快。本文描述了一种方法，该方法可根据MALDI光谱中阶梯峰的时间依赖性定量测定反应速率。类似的方法可用于机理研究。
