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Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer
Published on: August 2, 2018
Phosphopeptide elution times in reversed-phase liquid chromatography
Jeongkwon Kim1, Konstantinos Petritis, Yufeng Shen
1Environmental Molecular Science Laboratory, MSIN K8-98, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
Journal of Chromatography. A
|October 16, 2007
Summary
Phosphorylation significantly alters peptide elution times in liquid chromatography, with smaller peptides showing greater shifts. This finding aids in confidently identifying and confirming phosphopeptides.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Phosphorylation is a critical post-translational modification regulating numerous cellular processes.
- Accurate identification of phosphopeptides is essential for understanding signaling pathways.
- Reversed-phase liquid chromatography coupled with tandem mass spectrometry (RPLC-MS/MS) is a primary tool for phosphoproteomic analysis.
Purpose of the Study:
- To systematically investigate the impact of phosphorylation on peptide elution times.
- To analyze how peptide length influences phosphorylation-induced elution time shifts.
- To assess the predictive value of elution time changes for phosphopeptide identification.
Main Methods:
- High-resolution reversed-phase liquid chromatography (RPLC) coupled with tandem mass spectrometry (MS/MS).
- Analysis of 33 peptides and their corresponding phosphopeptides.
- Use of trifluoroacetic acid as an ion-pairing agent.
Main Results:
- Single phosphorylation caused elution time shifts ranging from -5.28 to +0.59 min.
- Phosphotyrosine peptides eluted significantly faster than phosphoserine or phosphothreonine peptides.
- Smaller peptides (<18 amino acids) exhibited larger elution time deviations compared to larger peptides (>=18 amino acids).
Conclusions:
- Phosphorylation generally decreases peptide elution time, but exceptions exist.
- Peptide length is a critical factor influencing the magnitude of elution time shifts.
- Understanding these elution time shifts improves the confidence of phosphopeptide identification and confirmation.
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