Uslan Travis, Quan Baiyi, Wang Ting-Yu, Pang Marion, Qiu Yanping, Chou Tsui-Fen
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, California 91125, United States.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
ACS Omega. 2025 Mar 6;10(10):10642-10652. doi: 10.1021/acsomega.4c11585. eCollection 2025 Mar 18.
Proteomic analysis plays an essential role in biology with several methodologies available for sample preparation and analysis. This study evaluates and compares various cell lysis and protein digestion protocols for bottom-up proteomics using HeLa S3 cells. We assessed two physical disruption methods to homogenize cells-sonication and BeatBox-alongside four digestion protocols. Two of them are lab-reagent strategies: urea-based and sodium deoxycholate (SDC)-based in-solution digestion, and two are commercially available kits: the EasyPep kit from Thermo Fisher Scientific and S-Trap from Protifi. Each method's efficacy was evaluated based on protein recovery, peptide yield, and number of unique proteins identified through LC-MS analysis. Our results indicate that while both sonication and the BeatBox (PreOmics Inc.) methods provided comparable protein recovery and coverage, the choice of digestion method had a much bigger impact on the amount of protein IDs found. SDC digestion yielded the highest protein and peptide counts, while S-Trap exhibited the most consistent peptide recovery. Conversely, EasyPep showed higher variability in peptide recovery, with a ±10% difference in the average peptide number. Each homogenization strategy and digestion method also yielded its own list of unique proteins. These results provide several lists of proteins for biologists to select from based on experimental needs and highlight the importance of choosing appropriate protocols for comprehensive proteomic analyses.
蛋白质组学分析在生物学中起着至关重要的作用，有多种方法可用于样品制备和分析。本研究评估并比较了使用HeLa S3细胞进行自下而上蛋白质组学的各种细胞裂解和蛋白质消化方案。我们评估了两种使细胞匀浆的物理破碎方法——超声处理和BeatBox，以及四种消化方案。其中两种是实验室试剂策略：基于尿素和基于脱氧胆酸钠（SDC）的溶液内消化，另外两种是市售试剂盒：赛默飞世尔科技的EasyPep试剂盒和Protifi的S-Trap。基于蛋白质回收率、肽产量以及通过液相色谱-质谱分析鉴定出的独特蛋白质数量，评估了每种方法的效果。我们的结果表明，虽然超声处理和BeatBox（PreOmics公司）方法提供了相当的蛋白质回收率和覆盖率，但消化方法的选择对发现的蛋白质ID数量有更大影响。SDC消化产生的蛋白质和肽数量最多，而S-Trap的肽回收率最稳定。相反，EasyPep的肽回收率变异性较高，平均肽数量相差±10%。每种匀浆策略和消化方法还产生了各自独特的蛋白质列表。这些结果为生物学家根据实验需求提供了几份可供选择的蛋白质列表，并突出了选择合适方案进行全面蛋白质组学分析的重要性。
