title: Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity.
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).
authors.affiliation: Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200, Copenhagen, Denmark.
authors.forename: Rosa R
authors.initials: RR
authors.lastname: Jersie-Christensen
authors.affiliation: Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200, Copenhagen, Denmark.
authors.forename: Abida
authors.initials: A
authors.lastname: Sultan
authors.affiliation: Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200, Copenhagen, Denmark. jesper.olsen@cpr.ku.dk.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019295:Computational Biology; D030562:Databases, Protein; D006367:HeLa Cells; D006801:Humans; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014025:Titanium / Q000737:chemistry; D057188:Workflow
chemical_list: D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium
keywords: Heated guanidinium chloride lysis; Mass spectrometry; Phosphopeptide enrich ment; Phosphoproteomics; Sample preparation