دورية أكاديمية
أعرض في EDS

Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads.

التفاصيل البيبلوغرافية
العنوان: Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads.
المؤلفون: Johnston HE; Signalling Programme, The Babraham Institute, Cambridge CB22 3AT, United Kingdom., Yadav K; Mass Spectrometry Facility, The Babraham Institute, Cambridge CB22 3AT, United Kingdom., Kirkpatrick JM; Proteomics STP, The Francis Crick Institute, London NW1 1AT, United Kingdom., Biggs GS; Proteomics STP, The Francis Crick Institute, London NW1 1AT, United Kingdom.; GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, Hertfordshire, United Kingdom., Oxley D; Mass Spectrometry Facility, The Babraham Institute, Cambridge CB22 3AT, United Kingdom., Kramer HB; Medical Research Council London Institute of Medical Sciences, Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom., Samant RS; Signalling Programme, The Babraham Institute, Cambridge CB22 3AT, United Kingdom.
المصدر: Analytical chemistry [Anal Chem] 2022 Jul 26; Vol. 94 (29), pp. 10320-10328. Date of Electronic Publication: 2022 Jul 18.
نوع المنشور: Journal Article; Research Support, Non-U.S. Gov't
اللغة: English
بيانات الدورية: Publisher: American Chemical Society Country of Publication: United States NLM ID: 0370536 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1520-6882 (Electronic) Linking ISSN: 00032700 NLM ISO Abbreviation: Anal Chem Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Washington, American Chemical Society.
مواضيع طبية MeSH: Proteome*/analysis , Proteomics*, Magnetic Phenomena ; Protein Aggregates ; Reproducibility of Results ; Solvents
مستخلص: Complete, reproducible extraction of protein material is essential for comprehensive and unbiased proteome analyses. A current gold standard is single-pot, solid-phase-enhanced sample preparation (SP3), in which organic solvent and magnetic beads are used to denature and capture protein aggregates, with subsequent washes removing contaminants. However, SP3 is dependent on effective protein immobilization onto beads, risks losses during wash steps, and exhibits losses and greater costs at higher protein inputs. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3 protein cleanup, capturing acetonitrile-induced protein aggregates by brief centrifugation rather than magnetism─with optional low-cost inert glass beads to simplify handling. SP4 recovered equivalent or greater protein yields for 1-5000 μg preparations and improved reproducibility (median protein R 2 0.99 (SP4) vs 0.97 (SP3)). Deep proteome profiling revealed that SP4 yielded a greater recovery of low-solubility and transmembrane proteins than SP3, benefits to aggregating protein using 80 vs 50% organic solvent, and equivalent recovery by SP4 and S-Trap. SP4 was verified in three other labs across eight sample types and five lysis buffers─all confirming equivalent or improved proteome characterization vs SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein cleanup and identifies that magnetic capture risks losses, especially at higher protein concentrations and among more hydrophobic proteins. SP4 offers a minimalistic approach to protein cleanup that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications─all while retaining the speed and compatibility of SP3.
References: J Proteome Res. 2017 Feb 3;16(2):889-897. (PMID: 28088865)
Mol Syst Biol. 2019 Dec;15(12):e9021. (PMID: 31885202)
Proteomics. 2014 May;14(9):1006-0. (PMID: 24678027)
J Biomol Tech. 2002 Mar;13(1):1-4. (PMID: 19498958)
Nat Methods. 2014 Mar;11(3):319-24. (PMID: 24487582)
Mol Cell Proteomics. 2017 Dec;16(12):2055-2068. (PMID: 28982716)
J Vis Exp. 2022 Feb 7;(180):. (PMID: 35188123)
Nucleic Acids Res. 2019 Jan 8;47(D1):D442-D450. (PMID: 30395289)
Nucleic Acids Res. 2017 Jan 4;45(D1):D1112-D1116. (PMID: 27789699)
J Mol Biol. 2015 Jan 30;427(2):478-90. (PMID: 25451785)
Annu Rev Biomed Eng. 2009;11:49-79. (PMID: 19400705)
J Proteome Res. 2010 Jan;9(1):444-50. (PMID: 20000691)
Nat Protoc. 2019 Jan;14(1):68-85. (PMID: 30464214)
Rapid Commun Mass Spectrom. 2018 Aug 30;32(16):1414-1424. (PMID: 29857350)
J Proteome Res. 2019 Jul 5;18(7):2915-2924. (PMID: 31137935)
Protein Sci. 1994 Nov;3(11):1975-83. (PMID: 7703844)
Anal Biochem. 1984 Apr;138(1):141-3. (PMID: 6731838)
J Proteome Res. 2018 Apr 6;17(4):1730-1740. (PMID: 29565595)
Mol Syst Biol. 2020 Jan;16(1):e9111. (PMID: 32129943)
Mol Cell Proteomics. 2020 Jan;19(1):209-222. (PMID: 31754045)
Sci Rep. 2016 Oct 07;6:34949. (PMID: 27713570)
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Mar 5;785(2):263-75. (PMID: 12554139)
BMC Bioinformatics. 2006 Jul 04;7:331. (PMID: 16817977)
J Chromatogr A. 2004 Jan 16;1023(2):317-20. (PMID: 14753699)
Mol Cell Proteomics. 2019 May;18(5):1027-1035. (PMID: 30833379)
Proteomics. 2017 Oct;17(20):. (PMID: 28271630)
J Proteome Res. 2017 Nov 3;16(11):4060-4072. (PMID: 28948796)
Anal Chem. 2009 May 1;81(9):3285-96. (PMID: 19323527)
Electrophoresis. 2010 Oct;31(21):3573-9. (PMID: 20967768)
J Proteome Res. 2020 May 1;19(5):2035-2042. (PMID: 32195589)
Nat Methods. 2009 May;6(5):359-62. (PMID: 19377485)
Mol Syst Biol. 2014 Oct 30;10:757. (PMID: 25358341)
Mol Cell Proteomics. 2021;20:100039. (PMID: 33476790)
Curr Eye Res. 1998 Nov;17(11):1074-81. (PMID: 9846626)
معلومات مُعتمدة: BB/P013384/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
المشرفين على المادة: 0 (Protein Aggregates)
0 (Proteome)
0 (Solvents)
تواريخ الأحداث: Date Created: 20220718 Date Completed: 20220727 Latest Revision: 20220803
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC9330274
DOI: 10.1021/acs.analchem.1c04200
PMID: 35848328
قاعدة البيانات: MEDLINE
الوصف
تدمد:1520-6882
DOI:10.1021/acs.analchem.1c04200