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PXD028736-2

PXD028736 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleSolvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads
DescriptionComplete, 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.
HostingRepositoryPRIDE
AnnounceDate2023-11-14
AnnouncementXMLSubmission_2023-11-14_08:53:25.823.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterGeorge Biggs
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListmonohydroxylated residue; iodoacetamide derivatized residue
InstrumentOrbitrap Fusion Lumos
Dataset History
RevisionDatetimeStatusChangeLog Entry
02021-09-24 05:00:57ID requested
12022-08-04 09:04:59announced
22023-11-14 08:53:26announced2023-11-14: Updated project metadata.
Publication List
Dataset with its publication pending
Keyword List
submitter keyword: proteomics,Solvent precipitation, sample preparation
Contact List
Rahul Samant
contact affiliationBabraham Institute Cambridge CB22 3AT
contact emailrahul.samant@babraham.ac.uk
lab head
George Biggs
contact affiliationThe Francis Crick Institute
contact emailgeorge.biggs@crick.ac.uk
dataset submitter
Full Dataset Link List
Dataset FTP location
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PRIDE project URI
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