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Reputable Mentor II
Reputable Mentor II
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
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‎10-15-2021 10:26 AM
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