Publication Information
Wolschin et al., 2005
No external accession available
Abstract
Plant Methods. 2005 Nov 1;1(1):9. doi: 10.1186/1746-4811-1-9.
Combining metal oxide affinity chromatography (MOAC) and selective mass
spectrometry for robust identification of in vivo protein phosphorylation sites.
Wolschin F(1), Weckwerth W.
Author information:
(1)Max Planck Institute of Molecular Plant Physiology, 14424 Potsdam, Germany.
weckwerth@mpimp-golm.mpg.de
BACKGROUND: Protein phosphorylation is accepted as a major regulatory pathway in
plants. More than 1000 protein kinases are predicted in the Arabidopsis
proteome, however, only a few studies look systematically for in vivo protein
phosphorylation sites. Owing to the low stoichiometry and low abundance of
phosphorylated proteins, phosphorylation site identification using mass
spectrometry imposes difficulties. Moreover, the often observed poor quality of
mass spectra derived from phosphopeptides results frequently in uncertain
database hits. Thus, several lines of evidence have to be combined for a precise
phosphorylation site identification strategy.
RESULTS: Here, a strategy is presented that combines enrichment of
phosphoproteins using a technique termed metaloxide affinity chromatography
(MOAC) and selective ion trap mass spectrometry. The complete approach involves
(i) enrichment of proteins with low phosphorylation stoichiometry out of complex
mixtures using MOAC, (ii) gel separation and detection of phosphorylation using
specific fluorescence staining (confirmation of enrichment), (iii)
identification of phosphoprotein candidates out of the SDS-PAGE using liquid
chromatography coupled to mass spectrometry, and (iv) identification of
phosphorylation sites of these enriched proteins using automatic detection of
H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the
corresponding MS2-fragment. The utility of this approach is demonstrated by the
identification of phosphorylation sites in Arabidopsis thaliana seed proteins.
Regulatory importance of the identified sites is indicated by conservation of
the detected sites in gene families such as ribosomal proteins and sterol
dehydrogenases. To demonstrate further the wide applicability of MOAC,
phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures.
CONCLUSION: A novel phosphoprotein enrichment procedure MOAC was applied to seed
proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the
method can easily be adapted to suit the sample of interest since it is
inexpensive and the components needed are widely available. Reproducibility of
the approach was tested by monitoring phosphorylation sites on specific proteins
from seeds and C. reinhardtii in duplicate experiments. The whole process is
proposed as a strategy adaptable to other plant tissues providing high
confidence in the identification of phosphoproteins and their corresponding
phosphorylation sites.
DOI: 10.1186/1746-4811-1-9
PMCID: PMC1295590
PMID: 16270910
