PXD028736-2

PXD028736 is an original dataset announced via ProteomeXchange.

Title	Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads
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.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:53:25.823.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	George Biggs
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2021-09-24 05:00:57	ID requested
1	2022-08-04 09:04:59	announced
⏵ 2	2023-11-14 08:53:26	announced	2023-11-14: Updated project metadata.

Dataset with its publication pending

submitter keyword: proteomics,Solvent precipitation, sample preparation

Rahul Samant
contact affiliation	Babraham Institute Cambridge CB22 3AT
contact email	rahul.samant@babraham.ac.uk
lab head
George Biggs
contact affiliation	The Francis Crick Institute
contact email	george.biggs@crick.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD028736
     1. Label: PRIDE project
     2. Name: Solvent Precipitation SP3 (SP4) enhances recovery for proteomics sample preparation without magnetic beads