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Abstract
The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.
Highlights
Phosphorylation is a rapid, dynamic, and reversible post-translational modification that regulates the diversity of protein functions [1,2,3]
We present an optimized protocol for label-free quantitative (LFQ) phosphoproteomics analysis of the whole-cell extract of the fission yeast S. pombe
Our LFQ phosphoproteomics workflow is based on Fe-immobilized metal affinity chromatography (IMAC) phosphopeptide enrichment, followed by porous graphitic carbon (PGC) and strong anion exchange (SAX)
Summary
Phosphorylation is a rapid, dynamic, and reversible post-translational modification that regulates the diversity of protein functions [1,2,3]. For a given amino acid, the degree of phosphorylation, which reflects how many copies of a protein are phosphorylated at a particular amino acid at a given time point, might represent a threshold that distinguishes the relevant phosphorylation from the non-functional phosphorylation events This makes the identification and the analysis of biologically relevant protein phosphorylation challenging [4,5,6]. Our LFQ phosphoproteomics workflow is based on Fe-IMAC phosphopeptide enrichment, followed by PGC and SAX fractionations of the phosphopeptides (Figure 1). The protocol can be parallelized with other phosphopeptide enrichment and fractionation strategies, which might further improve the identification of phosphopeptides Using this approach, we were able to detect 7079 annotated phosphosites and have identified an additional and so far non-annotated 1274 phosphosites, which represent a substantial addition to the phosphoproteome of the fission yeast S. pombe.
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