He Qiqing, Chen Qingjing, Wang Dongxue, He Fuchu
Department of Chemistry, School of Science, Southern University of Science and Technology, Shenzhen, China.
State Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China.
Bio Protoc. 2025 Jul 20;15(14):e5398. doi: 10.21769/BioProtoc.5398.
This manuscript details protocols for the ZnCl precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl at 100 mM and 50% methanol), ZASP depletes harsh detergents and impurities, such as sodium dodecyl sulfate (SDS), Triton X-100, and urea, at high concentrations in solution from protein solutions prior to trypsin digestion. It is a practical, robust, and cost-effective approach for proteomic sample preparation. It has been observed that 90.2% of the proteins can be recovered from lysates by incubating with an equal volume of ZPB at room temperature for 10 min. In 1 h of data-dependent acquisition (DDA) analysis on an Exploris 480, 4,037 proteins and 25,626 peptides were quantified from 1 μg of mouse small intestine proteins, reaching a peak of 4,500 proteins and up to 30,000 peptides with 5 μg of input. Additionally, ZASP outperformed other common sample preparation methods such as sodium deoxycholate (SDC)-based in-solution digestion, acetone precipitation, filter-aided sample preparation (FASP), and single-pot, solid-phase-enhanced sample preparation (SP3). It demonstrated superior performance in protein (4,456 proteins) and peptide identification (29,871 peptides), lower missing cleavage rates (16.3%), and high reproducibility (Pearson correlation coefficients of 0.96 between replicates) with similar protein distributions and cellular localization patterns. Significantly, the cost of ZASP per sample with 100 μg of protein as input is lower than 30 RMB, including the expense of trypsin. Key features • ZASP is a user-friendly method that enables efficient, sensitive, and cost-effective proteomic analysis in a common biochemistry lab through simple incubation and centrifugation. • ZASP enables 90% protein recovery from lysates with various trypsin and LC-MS-incompatible reagents by incubating at room temperature for 10 min. • ZASP enables unbiased proteomic analysis of diverse biological samples, including cells, optimal cutting temperature (OCT)-embedded tissues, and formalin-fixed paraffin-embedded (FFPE) tissues.
本手稿详细介绍了用于蛋白质组分析的氯化锌沉淀辅助样品制备（ZASP）方案。通过用ZASP沉淀缓冲液（ZPB，氯化锌终浓度为100 mM且甲醇浓度为50%）诱导蛋白质沉淀，ZASP在胰蛋白酶消化前从蛋白质溶液中去除了溶液中高浓度的苛刻去污剂和杂质，如十二烷基硫酸钠（SDS）、Triton X-100和尿素。它是一种实用、稳健且经济高效的蛋白质组样品制备方法。据观察，通过在室温下与等体积的ZPB孵育10分钟，可从裂解物中回收90.2%的蛋白质。在Exploris 480上进行1小时的数据依赖采集（DDA）分析时，从1μg小鼠小肠蛋白质中定量了4037种蛋白质和25626种肽段，输入5μg时蛋白质峰值达到4500种，肽段多达30000种。此外，ZASP优于其他常见的样品制备方法，如基于脱氧胆酸钠（SDC）的溶液内消化、丙酮沉淀、过滤辅助样品制备（FASP）和单罐、固相增强样品制备（SP3）。它在蛋白质（4456种蛋白质）和肽段鉴定（29871种肽段）方面表现出卓越性能，具有更低的错切率（16.3%）以及高重现性（重复之间的皮尔逊相关系数为0.96），且蛋白质分布和细胞定位模式相似。值得注意的是，以100μg蛋白质为输入时，每个样品的ZASP成本低于30元人民币，包括胰蛋白酶的费用。关键特性•ZASP是一种用户友好的方法，通过简单的孵育和离心，可在普通生物化学实验室中实现高效、灵敏且经济高效的蛋白质组分析。•通过在室温下孵育10分钟，ZASP能够从含有各种胰蛋白酶和与液相色谱-质谱不兼容试剂的裂解物中回收90%的蛋白质。•ZASP能够对包括细胞、最佳切割温度（OCT）包埋组织和福尔马林固定石蜡包埋（FFPE）组织在内的多种生物样品进行无偏倚的蛋白质组分析。
