Dataset Information

An optimized SP3 sample processing workflow for in-depth phos-phoproteomics and improved identification of multiply phosphory-lated peptides

ABSTRACT: Protein phosphorylation is a critical and ubiquitous post-translational modification (PTM) found across the kingdoms of life. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample prepara-tion methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological capabil-ities. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have revolutionized proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demon-strate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increas-ing the number nearly 2-fold compared to standard SP3 techniques. We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently underexplored, and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: leonard daly

LAB HEAD: Prof Claire Eyers

PROVIDER: PXD061013 | Pride | 2025-08-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	LD_211116_precipitation_phospho_Acetonitrile-1.mgf	Mgf
	LD_211116_precipitation_phospho_Acetonitrile-1.mzid	Mzid
	LD_211116_precipitation_phospho_Acetonitrile-1.raw	Raw
	LD_211116_precipitation_phospho_Acetonitrile-2.mgf	Mgf
	LD_211116_precipitation_phospho_Acetonitrile-2.mzid	Mzid

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Publications

An Optimized SP3 Sample Processing Workflow for In-Depth and Reproducible Phosphoproteomics.

Daly Leonard A LA Clarke Christopher J CJ Oswald Sally O SO Jankevics Andris A Brownridge Philip J PJ Scheltema Richard A RA Eyers Claire E CE

Journal of proteome research 20250717 8

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life and is critical for the regulation of protein function in health and disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an opti ...[more]

PMID: 40671572

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