Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag
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ABSTRACT:
SUBMITTER: Fujiwara Y
PROVIDER: S-EPMC8594809 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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 proteins, with subsequently washes allowing contaminant removal. However, SP3 is dependent on effective protein immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in protein recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief centrifugation—either with or without low-cost inert glass beads—as the means of aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1 — 5000 µg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs. 0.97 (SP3)). Deep proteome profiling (n=9,076) also demonstrated improved recovery by SP4 and a significant enrichment of membrane and low-solubility proteins vs. SP3. The effectiveness of SP4 was verified in three other labs, each confirming equivalent or improved proteome characterisation over SP3. This work suggests that protein precipitation is the primary mechanism of SP3, and reliance on magnetic beads presents protein losses, especially at higher concentrations and amongst hydrophobic proteins. SP4 represents an efficient and effective alternative to SP3, provides the option to omit beads entirely, and offers virtually unlimited scalability of input and volume—all whilst retaining the speed and universality of SP3.
2022-08-03 | PXD028768 | Pride
Project description: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 immobilisation onto beads, risks losses during wash steps, and experiences a drop-off in recovery at higher protein inputs. Magnetic beads may also contaminate samples and instruments, and become costly for larger-scale protein preparations. Here, we propose solvent precipitation SP3 (SP4) as an alternative to SP3, omitting magnetic beads and employing brief (5-minute) centrifugation—either with or without low-cost inert glass beads—as a means of acetonitrile-induced aggregated protein capture. SP4 recovered equivalent or greater protein yields for 1–5000µg preparations and improved reproducibility (median protein R2 0.99 (SP4) vs. 0.97 (SP3)). Deep proteome profiling revealed SP4 yielded 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 characterisation vs. SP3. With near-identical recovery, this work further illustrates protein precipitation as the primary mechanism of SP3 protein clean-up, and identifies that magnetic capture risks losses, especially at higher protein concentrations and amongst more hydrophobic proteins. SP4 offers a minimalistic approach to protein clean-up that provides cost-effective input scalability, the option to omit beads entirely, and suggests important considerations for SP3 applications—all whilst retaining the speed and compatibility of SP3.
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