| Autor: |
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. |
| Abstrakt: |
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. |
