Department of Chemistry, Chungnam National University, Daejeon, South Korea.
Rapid Commun Mass Spectrom. 2011 Jan 15;25(1):88-92. doi: 10.1002/rcm.4840.
The effect of vortex-induced vibration during tryptic digestion was investigated by applying different vibrational speeds (0, 600, 1200, or 2500 rpm) to digestion solutions for varying durations (10, 20, 30, 40, or 60 min) at two different incubation temperatures (25°C or 37°C). The most rapid digestion was observed with the highest vibrational speed and temperature. With the application of 2500 rpm at 37°C, the tryptic digestion of each of three standard proteins (cytochrome c, myoglobin, or bovine serum albumin) provided complete disappearance of the protein within 60 min, as determined by matrix-assisted laser desorption/ionization mass spectrometry. Compared to conventional overnight digestion, 60-min vortex-assisted tryptic digestion generated longer peptides, due primarily to the limited digestion time and provided better sequence coverages (89% vs. 78% for cytochrome c, 100% vs. 87% for myoglobin, and 38% vs. 26% for BSA). The longer peptides should be advantageous to analytical methods such as the middle-down approach that benefit from increased sequence coverage of proteins. Vortex-assisted tryptic digestion is expected to be a useful method for rapid tryptic digestion of proteins.
研究了在不同的孵育温度（25°C 或 37°C）下，用不同的振动速度（0、600、1200 或 2500 rpm）和不同的消化时间（10、20、30、40 或 60 min）对消化液施加涡旋振动的影响。在最高振动速度和温度下，消化速度最快。在 37°C 下应用 2500 rpm，三种标准蛋白质（细胞色素 c、肌红蛋白或牛血清白蛋白）中的每一种在 60 分钟内通过基质辅助激光解吸/电离质谱法确定完全消失，与传统的过夜消化相比，60 分钟涡旋辅助胰蛋白酶消化产生了更长的肽段，这主要是由于消化时间有限，并提供了更好的序列覆盖率（细胞色素 c 为 89%对 78%，肌红蛋白为 100%对 87%，BSA 为 38%对 26%）。较长的肽段应该有利于中间向下方法等分析方法，这些方法受益于蛋白质序列覆盖率的提高。涡旋辅助胰蛋白酶消化有望成为一种快速胰蛋白酶消化蛋白质的有用方法。
