on 09-26-201603:45 AM - edited on 10-15-202110:26 AM by Closed Account
Sergei I. Snovida1, Jae Choi1, Ryan Bomgarden1, Julian Saba2, Rosa Viner2, John C. Rogers1 International Hupo 2016 Purpose: The objective of this study is to demonstrate the utility and benefits of high-pH reversed-phase fractionation of phosphopeptides in MS-based phosphoproteomics workflows.
Methods: Lysates of K562 cells were digested with Lys-C and trypsin. Following the digestion, samples were enriched for phosphopeptides using Fe-NTA resin. Enriched phosphopeptides were fractionated in high-pH reversed-phased mode off-line with a Thermo Scientific™ Pierce™ High pH Reversed Phase Peptide Fractionation Kit using a bench top microcentrifuge. Absolute peptide quantities, including total post-enrichment yields and fractional distributions of enriched peptides, were determined using Thermo Scientific™ Pierce™ Quantitative Colorimetric Kit. LC-MS analysis was performed using Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer.
Results: The use of Thermo Scientific™ Pierce™ High pH Reversed Phase Peptide Fractionation Kits enables efficient, loss-free desalting of phosphopeptides following enrichment. High-pH reversed-phase fractionation of phosphopeptides leads to ~3-fold increase in phosphopeptide identifications relative to unfractionated samples.
1 Thermo Fisher Scientific, Rockford, IL, USA
2 Thermo Fisher Scientific, San Jose, Ca, USA