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

TY - CHAP

T1 - Simple and Reproducible Sample Preparation for Single-Shot Phosphoproteomics with High Sensitivity

AU - Jersie-Christensen, Rosa R

AU - Sultan, Abida

AU - Olsen, Jesper V

PY - 2016/1/1

Y1 - 2016/1/1

N2 - 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).

AB - 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).

U2 - 10.1007/978-1-4939-3049-4_17

DO - 10.1007/978-1-4939-3049-4_17

M3 - Book chapter

C2 - 26584931

SN - 978-1-4939-3048-7

T3 - Methods in molecular biology (Clifton, N.J.)

SP - 251

EP - 260

BT - Phospho-Proteomics

A2 - Stechow, Louise von

ER -