通过使用弱离子交换从蛋白质消化器中提取高效,低成本和高吞吐量的二甲基硫酸盐 (SDS)
Weikai Cao1, Phillip Y Chu2, Merlin L Bruening1,3
1Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States.
Journal of the American Society for Mass Spectrometry
|December 9, 2024
概括
一种新的,廉价的方法有效地使用弱离子交换 (WAX) 树脂从蛋白质样本中去除二硫酸盐 (SDS). 这种高通量技术对于精确的液体染色学-双重质谱 (LC-MS/MS) 蛋白质分析至关重要.
科学领域:
- 生物化学 生物化学
- 分析化学 分析化学
- 蛋白质组学是指蛋白质组学.
背景情况:
- 二硫酸盐 (SDS) 对于蛋白质变性和提取至关重要,但会干扰液体染色学-并联质谱学 (LC-MS/MS).
- 高SDS度会导致柱子过压,保留时间转移和LC-MS/MS中的信号抑制等问题.
- 有效的SDS去除对于蛋白质消化物的可靠LC-MS/MS分析至关重要.
研究的目的:
- 开发一种廉价,高通量的方法,从蛋白质消化器中去除SDS.
- 为了验证SDS去除方法的效率和回收.
- 评估该方法在测量血清等复杂生物矩阵中的蛋白质的实用性.
主要方法:
- 在96孔过板中利用弱离子交换 (WAX) 树脂来去除SDS.
- 经过加工的蛋白质消化剂具有不同的SDS度 (0.1-1%).
- 分析回收和量化trastuzumab在SDS移除后的尖小鼠血清中.
主要成果:
- 在不到3分钟的时间内,SDS度从0.1-0.4%降至<5ppm,从0.6-1%降至<100ppm.
- 对于未经修改的三性,达到约90%的恢复,对于化,达到约70%的恢复.
- 在小鼠血清中证明了优异的线性 (R2 = 0.9996) 和trastuzumab量化准确性.
结论:
- WAX树脂方法为LC-MS/MS之前的SDS去除提供了一种高效,具有成本效益的解决方案.
- 这种高通量技术确保了高回收率和精确的蛋白质定量.
- 该方法的可负担性和易于实施使其高度可转移到其他实验室.
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