ac502708m_si_001.xls (5.99 MB)
Highly Sensitive Phosphoproteomics by Tailoring Solid-Phase Extraction to Electrostatic Repulsion-Hydrophilic Interaction Chromatography
dataset
posted on 2015-02-03, 00:00 authored by Stefan Loroch, René
Peiman Zahedi, Albert SickmannIn the past decade, several strategies
for comprehensive phosphoproteome
analysis have been introduced. Most of them combine different phosphopeptide
enrichment techniques and require starting material in the milligram
range, as a consequence of their insufficient sensitivity. This limitation
impairs the applicability of phosphoproteomics to a wide variety of
clinical research, where sample material is highly limited. Here we
introduce a highly sensitive and easy-to-establish 2D bottom-up strategy
for microgram-scale phosphoproteomics, based on electrostatic repulsion–hydrophilic
interaction chromatography (ERLIC), a simple solid-phase extraction
step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS
analysis. With only 100 μg of tryptic digested, nonstimulated
HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500
nonredundant and highly confident phosphorylation sites (per replicate).
We assigned all phosphorylation sites to 3013 phosphoproteins, covering
the entire dynamic range from 107 down to a few copies
per cell. Compared to affinity-based-enrichment methods using Ti4+, our ERLIC-based strategy enriched considerably longer and
more acidic phosphopeptides and consequently, we identified 327 phosphorylated
C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS
render its future promising for microgram-scale-phosphoproteomics
in biological, biomedical, and clinical research.