Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry.
Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry.
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通过质谱法测量糖化血红蛋白 A1c 来评估微波辅助酶消化。
DOI：
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发表时间：
2005
影响因子：
2
通讯作者：
G. Myers
中科院分区：
文献类型：
--
作者：
H. Vesper;Luchuan Mi;Archibold Enada;G. Myers
蛋白质的酶促消化和通过质谱法分析所得肽是蛋白质组学以及临床和环境化学中的既定方法。长达数小时的消化时间阻碍了样品和结果的快速周转。定性应用表明，微波辐射大大缩短酶消化。然而，尚未评估其在定量应用方面的效用。在这项研究中，微波辅助酶消化血红蛋白在不同的温度，缓冲液浓度和消化时间进行了评估，并与传统的消化蛋白水解酶胰蛋白酶和Glu-C相比。采用微波辅助酶消化法对糖化血红蛋白HbA 1c进行分析，并与参考方法进行比较。使用胰蛋白酶，完全消化，在50 ℃，在20分钟内获得。在这些条件下，消化效率比传统的胰蛋白酶消化高20%。用Glu-C作为酶时未观察到这些效应，可能是因为与所用胰蛋白酶相比，Glu-C在高温下的稳定性降低。使用胰蛋白酶的优化微波辅助消化方法与使用Glu-C的HbA 1c参考方法的比较结果具有密切相关性（R2：0.996）。观察到0.33% HbA 1c的显著偏倚，微波辅助胰蛋白酶消化获得的值更高;这一发现可能是使用不同酶所致。这项研究表明，微波辅助酶消化可以大大减少消化时间到分钟，可用于定性和定量应用。
Enzymatic digestion of proteins and analysis of the resulting peptides by mass spectrometry is an established approach in proteomics and in clinical and environmental chemistry. The long digestion times of several hours prevent the fast turnover of samples and results. Qualitative applications showed that microwave radiation profoundly shortens enzymatic digestion. However, its usefulness for quantitative applications had not been assessed. In this study, the microwave-assisted enzymatic digestion of hemoglobin at different temperatures, buffer concentrations, and digestion times was assessed and compared with conventional digestion for the proteolytic enzymes trypsin and Glu-C. A microwave-assisted enzymatic digestion method optimized for digestion time and temperature was applied for the analysis of glycated hemoglobin HbA1c and compared with a reference method. Using trypsin, complete digestion was obtained at 50 degrees C within 20 min. Under these conditions, the digestion efficiency was 20% higher than with conventional trypsin digestion. These effects were not observed with Glu-C as enzyme, probably because of the decreased stability of Glu-C at elevated temperatures in comparison with the trypsin used. The comparison of the optimized microwave-assisted digestion method using trypsin with the reference method for HbA1c using Glu-C gave a close correlation in the results (R2: 0.996). A significant bias of 0.33% HbA1c was observed, with higher values obtained with the microwave-assisted tryptic digest; this finding might have resulted from the use of a different enzyme. This study showed that microwave-assisted enzymatic digestion can substantially reduce digestion times to minutes and can be used in qualitative as well as quantitative applications.
DOI：
10.1016/0006-291x(90)92080-j
发表时间：
1990-03-16
影响因子：
3.1
作者：
CHOWDHURY, SK;KATTA, V;CHAIT, BT
通讯作者：
CHAIT, BT