SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Peptide cleanup is crucial in bottom-up proteomics to remove contaminants from sample preparation.
• Carboxylate-modified paramagnetic particles offer advantages over traditional methods for detergent and polymer removal.
• Previous implementations faced reproducibility challenges, hindering widespread adoption.

Purpose of the Study:

• To systematically evaluate experimental parameters for carboxylate-modified paramagnetic particle-based peptide cleanup.
• To identify critical parameters for optimal performance and peptide recovery.
• To develop a reproducible and user-friendly standard operating protocol (SP2).

Main Methods:

• Systematic evaluation of key experimental parameters for carboxylate-modified paramagnetic particle usage.
• Development and validation of the SP2 protocol for peptide cleanup.
• Assessment of SP2 compatibility with phosphopeptides, glycopeptides, and automated liquid handling.

Main Results:

• Identified critical parameters for maximum performance and peptide recovery during particle-based cleanup.
• Developed SP2, a detailed and easy-to-use protocol for detergent and polymer removal.
• Demonstrated SP2's applicability to phosphopeptides and glycopeptides, and its compatibility with automated processing.

Conclusions:

• The SP2 protocol facilitates reproducible peptide cleanup and concentration for LC-MS workflows.
• SP2 overcomes previous reproducibility challenges, enabling wider laboratory implementation.
• This protocol is expected to benefit various proteomics applications, especially in core facilities dealing with diverse contaminants.