Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes

Abstract

BackgroundPhosphorylation is the most frequent post-translational modification made to proteins and may regulate protein activity as either a molecular digital switch or a rheostat. Despite the cornucopia of high-throughput (HTP) phosphoproteomic data in the last decade, it remains unclear how many proteins are phosphorylated and how many phosphorylation sites (p-sites) can exist in total within a eukaryotic proteome. We present the first reliable estimates of the total number of phosphoproteins and p-sites for four eukaryotes (human, mouse, Arabidopsis, and yeast).ResultsIn all, 187 HTP phosphoproteomic datasets were filtered, compiled, and studied along with two low-throughput (LTP) compendia. Estimates of the number of phosphoproteins and p-sites were inferred by two methods: Capture-Recapture, and fitting the saturation curve of cumulative redundant vs. cumulative non-redundant phosphoproteins/p-sites. Estimates were also adjusted for different levels of noise within the individual datasets and other confounding factors. We estimate that in total, 13 000, 11 000, and 3000 phosphoproteins and 230 000, 156 000, and 40 000 p-sites exist in human, mouse, and yeast, respectively, whereas estimates for Arabidopsis were not as reliable.ConclusionsMost of the phosphoproteins have been discovered for human, mouse, and yeast, while the dataset for Arabidopsis is still far from complete. The datasets for p-sites are not as close to saturation as those for phosphoproteins. Integration of the LTP data suggests that current HTP phosphoproteomics appears to be capable of capturing 70 % to 95 % of total phosphoproteins, but only 40 % to 60 % of total p-sites.

The serine/threonine phosphatases of apicomplexan parasites

“…Protein kinases and phosphatases oppositely regulate the most common post-translational modification, phosphorylation. A recent phosphoproteomics study estimated that nearly half of the total proteins from any of human, mouse or yeast are phosphoproteins (Vlastaridis et al, 2017). Therefore, it is reasonable to estimate that a comparable ratio of apicomplexan proteins is under the regulation of phosphorylation.…”

Phosphoproteomic Analyses Provide Insight into Molecular Mechanisms Underlying NETosis

“…Although several studies have clarified specific differences between NOX‐dependent and NOX‐independent NETosis, the underlying mechanisms of NETosis have not been fully elucidated. Phosphorylation is the most frequent post‐translational modification of proteins . In this study, Phosphoproteomics was employed to gain insight into the molecular mechanisms underlying NETosis and discover the common factors associated with both NOX‐dependent and NOX‐independent NETosis.…”

Protein Phosphorylation Dynamics: Unexplored Because of Current Methodological Limitations

“…Currently, sophisticated analytical methods (e.g., mass spectrometry, functional interaction traps, affinity chromatography) have taken great strides recently: they have facilitated the determination of the high‐throughput phospho‐proteome within many species and tissues, although the exact phospho landscape is impossible to determine, because of rapid turnover and the inevitable loss of material during the analytical extraction process. [ 2,24,25 ] It was found that 86% of all phosphorylated residues are serine, followed by 12% threonine and only 1.8% tyrosine. [ 5,24,25 ] These powerful technologies allowed the creation of public databases that report the exact and comprehensive phospho sites found in vivo for multiple species models.…”