Oyler Jonathan M, Tran Bao Q, Kilgour David P A
US Army Medical Research Institute of Chemical Defense, 8350 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010-5400, United States.
Technical Inspections Division (SAIG-TI), The US Army Inspector General Agency, 2530 Crystal Drive, Suite 12039, Arlington, Virginia 22202-3912, United States.
Anal Chem. 2021 Mar 30;93(12):5046-5053. doi: 10.1021/acs.analchem.0c04143. Epub 2021 Mar 17.
Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18-24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A toxin, enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.
基于自下而上质谱的蛋白质分析方法采用蛋白酶消化，常用于以高特异性和灵敏度鉴定和表征蛋白质。与预测数据库相比，方法性能通常通过序列覆盖能力和鉴定出的特征肽总数来衡量。目前常用的基于溶剂的消化方法存在局限性，包括消化时间长（18 - 24小时或更长），导致蛋白酶自溶，这也妨碍了自动化，降低了灵敏度，并增加了日内和日间性能变异性。本报告描述了一种简单的5分钟胰蛋白酶变性有机消化（DOD）方法的开发和验证，该方法用于自下而上的蛋白质鉴定和表征中的串联质谱分析。已针对选定的蛋白质毒素和诊断临床蛋白质靶点对其进行了评估，与其他基于溶液和酶固定化的方法相比，大大提高了消化性能。将该方法与两种目前使用的自下而上方法，即24小时滤膜辅助样品制备（FASP）和快速消化（1和4小时）方法进行了比较。用于比较这些方法的单一蛋白质包括蓖麻毒素轻链、蓖麻毒素重链、蓖麻毒素全毒素、血清型A毒素、肠毒素B、核糖核酸酶A和甲状腺球蛋白。在测试中，在所研究的蛋白质中，5分钟DOD消化方法的序列覆盖率为55%至100%，具有相对较高的重现性和精密度；结果优于或等于FASP方法的结果，与快速消化方法的结果相比有很大提高。重要的是，与FASP和快速消化的结果相比，DOD方法的日内和日间精密度有了很大提高。这些数据表明，与FASP和快速消化方法相比，DOD方法显著缩短了消化时间，同时保持或提高了检测和表征目标蛋白质的能力，并降低了胰蛋白酶消化的分析变异性，从而实现了明显更快、更精确的分析。
