Enhancing Proteome Coverage by Using Strong Anion-Exchange in Tandem with Basic-pH Reversed-Phase Chromatography for...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Proteomic studies require effective fractionation to maximize proteome coverage.
• Tandem Mass Tag (TMT) experiments, while powerful, necessitate fractionation for comprehensive protein identification.

Purpose of the Study:

• To develop and validate a simple, two-step peptide fractionation strategy combining strong anion-exchange (SAX) and basic pH reversed-phase (BPRP) chromatography.
• To improve proteome coverage and identify more unique peptides in TMT-based proteomic analyses.

Main Methods:

• A novel two-step peptide fractionation protocol using SAX spin columns followed by BPRP fractionation.
• Application of the strategy to a TMTpro18-plex experiment with nine human cell lines.
• Comparison of protein and peptide quantification between SAX-partitioned and BPRP-only fractionation datasets.

Main Results:

• The combined SAX-partition and BPRP fractionation strategy identified 10% more proteins and 20% more unique peptides compared to BPRP alone.
• Noticeable differences in peptide charge and isoelectric point distribution were observed between SAX partitions.
• Similar overall protein and peptide quantification numbers were achieved across different fractionation approaches.

Conclusions:

• The proposed SAX-partition followed by BPRP fractionation is an effective method to enhance proteome coverage in TMT-based proteomics.
• This strategy offers a valuable improvement for deep proteome profiling and biomarker discovery.
• An online resource of protein abundance profiles across nine human cell lines is provided.