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Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity

  • Rosa R. Jersie-Christensen
  • Abida Sultan
  • Jesper V. Olsen
Part of the Methods in Molecular Biology book series (MIMB, volume 1355)

Abstract

The traditional sample preparation workflow for mass spectrometry (MS)-based phosphoproteomics is time consuming and usually requires multiple steps, e.g., lysis, protein precipitation, reduction, alkylation, digestion, fractionation, and phosphopeptide enrichment. Each step can introduce chemical artifacts, in vitro protein and peptide modifications, and contaminations. Those often result in sample loss and affect the sensitivity, dynamic range and accuracy of the mass spectrometric analysis. Here we describe a simple and reproducible phosphoproteomics protocol, where lysis, denaturation, reduction, and alkylation are performed in a single step, thus reducing sample loss and increasing reproducibility. Moreover, unlike standard cell lysis procedures the cell harvesting is performed at high temperatures (99 °C) and without detergents and subsequent need for protein precipitation. Phosphopeptides are enriched using TiO2 beads and the orbitrap mass spectrometer is operated in a sensitive mode with higher energy collisional dissociation (HCD).

Key words

Phosphoproteomics Phosphopeptideenrichment Sample preparation Heated guanidinium chloride lysis Mass spectrometry 

Notes

Acknowledgements

The authors would like to thank members of the Proteomics Program at the Novo Nordisk Foundation Center for Protein Research (CPR) for critical input on the protocol. Work at CPR is funded in part by a generous donation from the Novo Nordisk Foundation (Grant number NNF14CC0001).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Rosa R. Jersie-Christensen
    • 1
  • Abida Sultan
    • 1
  • Jesper V. Olsen
    • 1
  1. 1.Proteomics Program, Novo Nordisk Foundation Center for Protein ResearchFaculty of Health and Medical Sciences, University of CopenhagenCopenhagenDenmark

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