Biology Reference
In-Depth Information
Chapter 42
Tandem Metal-Oxide Affi nity Chromatography
for Enhanced Depth of Phosphoproteome Analysis
Gerold J. M. Beckers , Wolfgang Hoehenwarter , Horst Röhrig ,
Uwe Conrath , and Wolfram Weckwerth
Abstract
In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In
recent years, phosphoproteomics has become a powerful tool to study protein phosphorylation because it
allows unbiased localization, and site-specifi c quantifi cation, of in vivo phosphorylation of hundreds of
proteins in a single experiment. A common strategy to identify phosphoproteins and their phosphorylation
sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates
followed by mass spectrometry. However, despite the high sensitivity of modern mass spectrometers the
large dynamic range of protein abundance and the transient nature of protein phosphorylation remained
major pitfalls in MS-based phosphoproteomics. Tandem metal-oxide affi nity chromatography (MOAC)
represents a robust and highly selective approach for the identifi cation and site-specifi c quantifi cation of low
abundant phosphoproteins that is based on the successive enrichment of phosphoproteins and -peptides.
This strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using
Al(OH)
3
-based MOAC, tryptic digestion of enriched phosphoproteins followed by TiO
2
-based MOAC of
phosphopeptides. Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and
phosphopeptides and, thus, allows probing of the phosphoproteome to unprecedented depth.
Key words
Phosphoproteomics, Tandem-MOAC, LC-MS, Orbitrap
1
Introduction
Reversible protein phosphorylation is a multifunctional posttrans-
lational modifi cation which serves to regulate enzyme catalytic
activity, localization, stability, and interactions. With 2-4 % of
eukaryotic proteomes accounting for kinases and phosphatases, it
is estimated that one-third of all proteins are phosphorylated.
Current advances for determining the phosphosites, stoichiome-
tries, and functional roles of each of these in vivo phosphorylations
Gerold J. M. Beckers and Wolfgang Hoehenwarter contributed equally to this work.
