Devi Shobha, Wu Bo-Hung, Chu Pei-Yu, Liu Yue-Pei, Wu Hsin-Lin, Ho Yen-Peng
Department of Chemistry, National Dong Hwa University, Hualien, Taiwan.
Electrophoresis. 2017 Feb;38(3-4):429-440. doi: 10.1002/elps.201600392. Epub 2016 Nov 15.
The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.
系统研究了微波辐照对溶液中消化过程及蛋白质检测限的影响。采用基质辅助激光解吸电离质谱（MALDI-MS）研究了在50℃微波加热条件下，各种蛋白质经胰蛋白酶消化产生的多种肽段的动力学过程。本研究还使用液相色谱-质谱联用（LC-MS）考察了在50℃微波加热及不同时间间隔（1、5和30分钟）下，单个蛋白质的检测限和消化完整性。我们得出结论，如果没有漏切的肽段决定检测限，传统消化将获得更好的检测限。如果带有漏切位点且强度较大的肽段在非常早期阶段（即少于1分钟）形成，并且在整个消化过程中不再进一步消化，那么微波加热和传统加热的检测限可能没有差异。比较了在传统条件和短时间（微波）条件下大肠杆菌裂解物的消化情况。在加热时间少于5分钟时，传统加热条件下鉴定出的蛋白质数量超过了微波加热条件下的数量。总体结果表明，微波辅助消化并不完全。尽管序列覆盖率可能更好，但检测限可能比传统加热条件下更差。
