Related Experiment Video
Updated: May 30, 2026
09:16
A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues
Published on: May 4, 2022
Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis.
Scott B Ficarro1, Yi Zhang, Marlene J Carrasco-Alfonso
1Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Molecular & Cellular Proteomics : MCP
|July 27, 2011
Summary
Novel multidimensional fractionation strategies enhance phosphoproteomics data production. A three-dimensional reversed-phase-strong anion exchange-reversed-phase (RP-SAX-RP) platform offers superior separation and identification of phosphopeptides.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Global phosphoproteomics is crucial for understanding cell signaling.
- Current phosphopeptide enrichment and fractionation methods limit large-scale data production.
Purpose of the Study:
- To develop and evaluate novel multidimensional fractionation strategies for phosphopeptide analysis.
- To improve the efficiency and scope of phosphoproteomics studies.
Main Methods:
- Exploration of two novel multidimensional fractionation strategies: 2D RP-RP with ion pairing agents and 3D RP-SAX-RP.
- Utilizing aliphatic ion pairing agents for enhanced phosphopeptide retention.
- Implementing strong anion exchange (SAX) as a second dimension in a 3D RP-SAX-RP setup.
Main Results:
- The 3D RP-SAX-RP platform demonstrated superior performance in separation peak capacity.
- This 3D approach identified more unique phosphopeptide sequences per microgram of cell lysate.
- The RP-SAX-RP platform showed orthogonal separation, high peak capacity, and efficient retention of phosphorylated peptides.
Conclusions:
- The integrated RP-SAX-RP platform enables automated, online LC-MS analysis of phosphopeptides.
- This method is reproducible and applicable to diverse biological samples, including leukemia models and activated T-cells.
