Combining Metabolic 15N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome
In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite 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. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118–124, 2012).
Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen (15N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of 15N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369–380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope (15N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while 15N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.
Key wordsPhosphoproteomics Tandem MOAC Metabolic labeling LC-MS
We thank present and former colleagues of our labs, especially Dennis Hopkins, Petra Majovsky, Bastian Minkenberg, Matthias Nagler, and Ella Nukarinen, who contributed to the development and optimization of the strategies described in this manuscript.
- 6.Arsova B, Kierszniowska S, Schulze WX (2012) The use of heavy nitrogen in quantitative proteomics experiments in plants. Trends Plant Sci 17:102–112Google Scholar