ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis
This manuscript details protocols for the ZnCl2 precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl 2 at 100 mM and 50% methanol), ZASP depletes harsh detergents and impurities, su...
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Bio-protocol LLC
2025-07-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=5398&type=0 |
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| author | Qiqing He Qingjing Chen Dongxue Wang Fuchu He |
| author_facet | Qiqing He Qingjing Chen Dongxue Wang Fuchu He |
| author_sort | Qiqing He |
| collection | DOAJ |
| description | This manuscript details protocols for the ZnCl2 precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl 2 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. |
| format | Article |
| id | doaj-art-443ae426863142ba9c367a2b2e622569 |
| institution | DOAJ |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-443ae426863142ba9c367a2b2e6225692025-08-20T02:46:00ZengBio-protocol LLCBio-Protocol2331-83252025-07-01151410.21769/BioProtoc.5398ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic AnalysisQiqing He0Qingjing Chen1Dongxue Wang2Fuchu He3Department of Chemistry, School of Science, Southern University of Science and Technology, Shenzhen, ChinaState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China, International Academy of Phronesis Medicine, Guangzhou, ChinaState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, ChinaInternational Academy of Phronesis Medicine, Guangzhou, China, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, ChinaInternational Academy of Phronesis Medicine, Guangzhou, China, Research Unit of Proteomics Driven Cancer Precision Medicine, Chinese Academy of Medical Sciences, Beijing, ChinaDepartment of Chemistry, School of Science, Southern University of Science and Technology, Shenzhen, ChinaState Key Laboratory of Medical Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China, International Academy of Phronesis Medicine, Guangzhou, China, Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou, China, Research Unit of Proteomics Driven Cancer Precision Medicine, ChinesThis manuscript details protocols for the ZnCl2 precipitation-assisted sample preparation (ZASP) for proteomic analysis. By inducing protein precipitation with ZASP precipitation buffer (ZPB, final concentration of ZnCl 2 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.https://bio-protocol.org/en/bpdetail?id=5398&type=0 |
| spellingShingle | Qiqing He Qingjing Chen Dongxue Wang Fuchu He ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis Bio-Protocol |
| title | ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis |
| title_full | ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis |
| title_fullStr | ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis |
| title_full_unstemmed | ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis |
| title_short | ZnCl2 Precipitation-Assisted Sample Preparation for Proteomic Analysis |
| title_sort | zncl2 precipitation assisted sample preparation for proteomic analysis |
| url | https://bio-protocol.org/en/bpdetail?id=5398&type=0 |
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