Spin column-based peptide fractionation alternatives for streamlined tandem mass tag (SL-TMT) sample processing

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Deep proteome coverage is crucial for multiplexing experiments.
• Traditional liquid chromatography (LC) fractionation is a bottleneck, limiting throughput.
• Existing methods require dedicated LC systems, posing cost and accessibility challenges.

Purpose of the Study:

• To introduce and evaluate spin column-based peptide fractionation methods.
• To assess the efficiency of strong anion exchange (SAX) and high-pH reversed phase (HPRP) spin columns for fractionation.
• To demonstrate the utility of these methods for achieving deep proteome coverage in high-throughput applications.

Main Methods:

• Peptide fractionation using SAX spin columns.
• Peptide fractionation using HPRP spin columns.
• Combined SAX and HPRP spin column fractionation.
• LC-MS/MS analysis for deep proteome quantification.

Main Results:

• Achieved deep proteome coverage with over 8000 quantified proteins per experiment.
• Demonstrated successful fractionation of multiplexed samples simultaneously.
• Showcased proteome alterations in human cell lines treated with ubiquitin-proteasome system inhibitors.

Conclusions:

• Spin column-based peptide fractionation is a viable, high-throughput alternative to LC systems.
• These methods are cost-effective and accessible for laboratories lacking dedicated LC instruments.
• The strategy is suitable for deep proteome analysis and screening applications.