Life Sciences Mass Spectrometry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.
J Chromatogr A. 2010 Jan 1;1217(1):57-64. doi: 10.1016/j.chroma.2009.11.011. Epub 2009 Nov 10.
Accelerated tryptic digestion of a therapeutic protein including microwave irradiation and thermal transfer by convection at 60 degrees C and 37 degrees C was investigated. An analytical setup was devised to follow the protein digestion rate using 1D gel electrophoresis and liquid chromatography coupled a triple quadrupole linear ion trap mass spectrometer. The formation kinetic of its tryptic peptides was monitored in the selected monitoring mode (LC-SRM/MS). Different digestion end points (e.g. 2, 5, 10, 15, 30 and 60min) as well as an overnight digestion were tested using a therapeutic human monoclonal antibody (mAb) with the goal of its LC-SRM/MS quantification in human plasma. The peptides from the human mAb were generated at different rates and were classified into three categories: (1) the fast forming peptides, (2) the slow forming peptides and (3) the peptides degrading over time. For many monitored peptides, a heating temperature of 37 degrees C with a 750rpm mixing applied for at least 30min provided equivalent results to microwave-assisted digestion and generally allowed the achievement of an equivalent peptide concentration as an overnight digestion carried out at 37 degrees C. The disappearance of the protein of the heavy and light chains can be monitored by 1D gel electrophoresis but was found not to be representative of the final tryptic peptide concentrations. For quantitative purposes a stable isotope labeled version ((13)C(4), (15)N(1)) of the therapeutic protein was used. The labeled protein as internal standard was found to be very efficient to compensate for incomplete digestion or losses during sample preparation.
研究了包括微波辐射和对流热传递在内的 60°C 和 37°C 条件下治疗性蛋白的加速胰蛋白酶消化。设计了一种分析装置，使用 1D 凝胶电泳和液相色谱-三重四极杆线性离子阱质谱联用仪来跟踪蛋白消化速率。在选定的监测模式（LC-SRM/MS）下监测其胰蛋白酶肽的形成动力学。使用治疗性人源单克隆抗体（mAb）测试了不同的消化终点（例如 2、5、10、15、30 和 60min）和过夜消化，目的是对其进行 LC-SRM/MS 定量分析。不同的 mAb 肽以不同的速率生成，并分为三类：（1）快速形成肽，（2）缓慢形成肽和（3）随时间降解的肽。对于许多监测肽，在 37°C 下加热并以 750rpm 混合至少 30min 的条件下提供了与微波辅助消化相当的结果，并且通常允许实现与在 37°C 下进行的过夜消化相当的肽浓度。通过 1D 凝胶电泳可以监测重链和轻链蛋白的消失，但发现它不能代表最终胰蛋白酶肽浓度。为了定量目的，使用了治疗性蛋白的稳定同位素标记版本（(13)C(4)，(15)N(1)）。作为内标使用的标记蛋白被发现对于补偿不完全消化或样品制备过程中的损失非常有效。
