Department of Chemistry, Chungnam National University, Yuseong-Gu, Daejeon, South Korea.
Anal Biochem. 2011 Jul 1;414(1):125-30. doi: 10.1016/j.ab.2011.02.026. Epub 2011 Feb 23.
The effects of temperature on ultrasound-assisted tryptic protein digestion were comprehensively investigated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Three standard proteins, cytochrome c, myoglobin, and bovine serum albumin, were digested at 4°C (ice), room temperature (20-25), 37, and 55°C for 0 s, 30s, 1 min, and 5 min, in an ultrasonic bath. We found that the number of identified peptides generally increased with increasing temperature or digestion time. Compared with conventional overnight digestion at 37°C without ultrasonication, digestions performed under ultrasonication generally produced more peptides under most of the above listed conditions, mainly due to miscleaved peptides. Tryptic digestions were also performed under all the conditions evaluated without using ultrasound, where the most significant improvement with the application of ultrasound in terms of sequence coverage and the number of identified peptides was observed at 4°C, followed by room temperature, and 37°C, while no improvement was observed at 55°C with the application of ultrasound, which may be due to the fact that the current experiments were performed in an ultrasonic bath.
采用基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF MS）全面研究了温度对超声辅助胰蛋白酶蛋白消化的影响。在超声浴中，将 3 种标准蛋白（细胞色素 c、肌红蛋白和牛血清白蛋白）在 4°C（冰）、室温（20-25°C）、37°C 和 55°C 下分别于 0 s、30 s、1 min 和 5 min 进行消化。我们发现，鉴定的肽的数量通常随温度或消化时间的增加而增加。与常规的在 37°C 下不进行超声作用的过夜消化相比，在大多数上述条件下，超声作用下的消化通常会产生更多的肽，这主要是由于肽的错误切割。在评估的所有条件下都未使用超声进行胰蛋白酶消化，在所有条件下应用超声时，在 4°C 下观察到序列覆盖率和鉴定肽数量的最大改善，其次是室温，37°C 下也有改善，而在 55°C 下应用超声则没有改善，这可能是因为当前实验是在超声浴中进行的。
