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Revisiting Protein Reversed-Phase Chromatography for Bottom-Up Proteomics
Shunsuke Takagi1,2, Nobuyuki Suzuki2, Yasushi Ishihama1,3
1Department of Molecular Systems BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Journal of Proteome Research
|September 18, 2024
Summary
Protein reversed-phase chromatography (Prot-RP) is an effective prefractionation method for bottom-up proteomics. It offers higher protein recovery than SDS-PAGE and identifies more protein termini and isoform-specific peptides than peptide RP.
Area of Science:
- Proteomics
- Chromatography
- Biochemistry
Background:
- Bottom-up proteomics requires efficient prefractionation strategies.
- Traditional methods like SDS-PAGE and peptide-level chromatography have limitations.
Purpose of the Study:
- To evaluate protein reversed-phase chromatography (Prot-RP) as a prefractionation step for bottom-up proteomics.
- To compare Prot-RP with SDS-PAGE and high-pH peptide RP (Pept-RP).
Main Methods:
- Utilized state-of-the-art RP columns for protein separation.
- Applied Prot-RP, SDS-PAGE, and Pept-RP to cell lysates.
- Analyzed fractions using mass spectrometry for proteoform profiling.
Main Results:
- Prot-RP demonstrated comparable fraction overlap to SDS-PAGE but with 2-fold higher protein recovery.
- Prot-RP showed slightly larger fraction overlap than Pept-RP.
- Prot-RP identified more protein termini and isoform-specific peptides compared to Pept-RP.
Conclusions:
- Protein RP prefractionation is a valuable technique for enhancing bottom-up proteomics.
- Prot-RP combined with advanced mass spectrometry is effective for proteoform profiling in cellular samples.
