Comparative phosphoproteomics reveals evolutionary and functional conservation of phosphorylation across eukaryotes
© Boekhorst et al.; licensee BioMed Central Ltd. 2008
Received: 08 July 2008
Accepted: 01 October 2008
Published: 01 October 2008
Reversible phosphorylation of proteins is involved in a wide range of processes, ranging from signaling cascades to regulation of protein complex assembly. Little is known about the structure and evolution of phosphorylation networks. Recent high-throughput phosphoproteomics studies have resulted in the rapid accumulation of phosphopeptide datasets for many model organisms. Here, we exploit these novel data for the comparative analysis of phosphorylation events between different species of eukaryotes.
Comparison of phosphoproteomics datasets of six eukaryotes yields an overlap ranging from approximately 700 sites for human and mouse (two large datasets of closely related species) to a single site for fish and yeast (distantly related as well as two of the smallest datasets). Some conserved events appear surprisingly old; those shared by plant and animals suggest conservation over the time scale of a billion years. In spite of the hypothesized incomprehensive nature of phosphoproteomics datasets and differences in experimental procedures, we show that the overlap between phosphoproteomes is greater than expected by chance and indicates increased functional relevance. Despite the dynamic nature of the evolution of phosphorylation, the relative overlap between the different datasets is identical to the phylogeny of the species studied.
This analysis provides a framework for the generation of biological insights by comparative analysis of high-throughput phosphoproteomics datasets. We expect the rapidly growing body of data from high-throughput mass spectrometry analysis to make comparative phosphoproteomics a powerful tool for elucidating the evolutionary and functional dynamics of reversible phosphorylation.
Post-translational modifications play important roles in a wide range of cellar functions. Reversible phosphorylation has been studied extensively and is known to influence protein function by changing protein-protein binding properties, activity, stability, and spatial organization . Phosphorylation plays a key role in signal transduction cascades  and allows the fine tuning of protein complex assembly . It is estimated that about one-third of all proteins in eukaryotic cells are phosphorylated at any given time .
Recent developments in high-throughput phosphoproteomics studies have resulted in the availability of phosphopeptide datasets for many model organisms. As a result, tools for the comparison of phosphoproteomes are emerging . Although these high-throughput datasets do not capture all phosphorylated peptides of a species under a given condition, large advances in enrichment strategies and mass spectrometry techniques have been made in the past few years, and studies comparing partial phosphoproteomes are emerging . Even though both the incomprehensive nature of the data as well as differences in experimental procedures complicate comparative analysis, we can now start to exploit these data. Comparative analysis of phosphoproteomics data could increase our understanding of phosphorylation and the evolution of the phosphorylation network as a systems level property.
Not only do comparative analyses aid in elucidating the evolution of phosphorylation, but they also are a powerful tool with which to improve function prediction from sometimes noisy high-throughput datasets. For example, the use of conserved gene order has been shown to be a much stronger signal for protein function prediction than the order of genes in a single genome [6–8]. Similarly, the conservation of co-expression has been shown to aid function prediction from microarray data [9, 10].
In this study we perform comparative analysis of phosphorylation events in eukaryotes. Our aim is to determine whether the quality of the data is sufficient to detect functionally significant overlap between high-throughput phosphoproteomics datasets, and to identify an evolutionarily significant pattern in this overlap. To address these questions, we compare recent high-throughput phosphoproteomics datasets of human, mouse, zebra fish, fruit fly, yeast, and plant. We determine the overlap between these datasets and show that this overlap is statistically, functionally, and evolutionarily relevant.
Measuring the overlap in phosphoproteomes
Number of query phosphorylation sites with at least one conserved site in the target species
The overlap between phosphoproteomics sets is significant
In both a scenario in which the rate of evolution of reversible phosphorylation is so high that the species are too diverged to detect real homologous phosphosites, and when species completely re-wire their phosphoproteome after speciation, chance alone would result in a certain amount of overlap. We thus randomized for every protein in the datasets the positions of the phosphorylated residues across 1,000 trials and computed the average overlap. Note that this is a conservative null model, because it assumes that different species phosphorylate the same protein, whereas cases have been described in which different species use phosphorylation of different proteins for the regulation of the assembly of homologous protein complexes . The observed overlap is larger than the average random overlap for almost all species comparisons (Table 2), strongly suggesting that the observed overlap is the result of significant evolutionary conservation.
Number of sites found in three or more different species
Three different speciesa
Four different species
Five different species
Relative overlap between phosphoproteomics data sets contains a strong evolutionary signal
Two independent tests suggest that the phosphorylation overlap is quantitatively significant. As a next step we tested for qualitative relevance by searching for a possible evolutionary pattern in the conservation of phosphoproteomics datasets. Specifically, we wondered whether a purported dynamic system level property such as the phosphorylation repertoire reflects the species phylogeny. However, interpreting the relative differences in overlap is far from trivial, because a myriad of both biological and technical factors, ranging from the sensitivity of the mass spectrometry analysis to experimental conditions under which phosphoproteomes were sampled, convolute a potential signal.
Low-throughput experiments as a golden standard and conserved phosphosites and protein function
Conservation in sequence and gene order generally has functional meaning . Low-throughput experiments are in general considered to be more reliable than high-throughput experiments, because they tend to be more suited to controls and validation. Several databases collect experimental data on reversible phosphorylation, for example Phospho.ELM  and Phosida . Of all of the phosphosites in the human dataset, 2.5% have also been observed by a low-throughput experiment in the Phospho.ELM database; for the mouse dataset this is 2.0%. In contrast, 4.8% of the conserved sites in human and 4.2% of the conserved sites in mouse have been measured using low-throughput techniques, a significant increase (χ2 test P < 0.0001). This observation shows that putative phosphorylation events with homologs in other high-throughput experiments are less likely to be false positives. This increase in reliability suggests that the overlap between phosphoproteomics datasets could be used as a tool with which to assess the reliability of putative phosphosites identified in high-throughput experiments, similar to the use of comparative methods for improving reliability of interactomes .
Phosphorylation events identified in a single high-thoughput experiments are known to cluster outside globular domains, as meausured by PFAM . Of the events we analyzed, 15% are found inside a domain predicted using domain predictions from the PFAM database . When we only consider conserved phosphorylation events, this shows a slight increase to 17%. The similar percentage shows that the low occurrence of phosphoryalation in known globular domains holds true for evolutionarily conserved events, and hence is not the result of the presence of spurious phosphorylations in unconfirmed high-throughput data.
Both the incomprehensive nature of high-throughput phosphoproteomics experiments as well as idiosyncrasies of the experimental pipelines used by different laboratories complicate the comparison of high-throughput phosphoproteomics datasets. In addition, the data we are comparing result from experiments designed with different biological questions in mind; the plant experiment, for example, focuses on the phosphorylation of membrane associated proteins from cells grown in culture , whereas the mouse experiment uses protein extract from homogenized liver tissue . All of these differences will undoubtedly introduce dissimilarities in the observed phosphoproteomes that do not reflect the evolutionary changes in phosphorylation networks between the different species, making the overlap that we found a minimal estimate. Randomization trials, functional bias in highly conserved phosphorylation events, and the relative differences in overlap between the six high-throughput phosphoproteomics datasets all suggest the overlap between these datasets to be biologically relevant, and we successfully identified the evolutionary signal in this overlap. We find a number of phosphorylation events that are likely to predate the evolutionary split between plants and animals. These sites thus appear to be ancient in origin, which is perhaps surprising, given that phosphorylation is thought to be a subtle regulatory mechanism.
Our work suggests that our understanding of reversible phosphorylation can be increased by comparing the results of high-throughput phosphoproteomics analysis with those from large-scale in vitro phosphorylation assays (for example [24, 25]) or computationally predicted phosphoproteomes. In the current setup (comparing different mass spectrometry based high-throughput phosphoproteomics datasets), experimental idiosyncrasies already loom large over any comparison; hence, we did not include such datasets in this study. However, because we have now shown that the overlap is biologically significant, this restraint can be relaxed; comparative analysis in fact enables the use of the ever-increasing amount of data on phosphorylation obtained by high-throughput mass spectrometry experiments that were not designed specifically for this particular purpose.
Previous studies have described the conservation across multiple species of amino acid residues that are known to be phosphorylated in a specific organism  and have studied the conservation of the phosphorylation events themselves on a small scale (for example ). PhosphoBlast  provides a powerful tool with which to compare (phosphorylated) peptides, illustrated by the authors by comparing human and mouse phosphopeptide datasets. These studies revealed a relatively high conservation of amino acid residues that are known to be phosphorylated in one or more phosphoproteomics experiments, and identified a substantial overlap between the phosphoproteomes of different species. We extend this observation to larger evolutionary distances and show that the overlap is statistically, functionally, and evolutionarily relevant. These insights can applied, for example, to discriminating between noise and real phosphorylation events in high-throughput mass spectrometry experiments (analogous to the use of conserved gene order in the evaluation of BLAST significance scores ).
The presence of functionally and evolutionarily significant overlap between high-throughput phosphoproteomics experiments allows the use of comparative phosphoproteomics in the prediction and evaluation of phosphorylation networks, similar to the established use of comparative genomics and transcriptomics in the elucidation of protein functions and biological networks. We expect the rapidly growing amount of data from high-throughput mass spectrometry analysis to make comparative phosphoproteomics a powerful tool in predicting, evaluating, and understanding reversible phosphorylation.
Materials and methods
Table 1 lists the datasets compared in this study. Because our comparison of high-throughput datasets is already complicated by many factors, ranging from the incomprehensive nature of the data to differences in experimental procedures, we made an effort to keep putative false-positive phosphorylation sites from further confounding the analysis. We used criteria for filtering the input data that in many cases are more stringent than the criteria used in the original publications. Each dataset was preprocessed by removing all phosphopeptides with ambiguous sites (phosphogroups that could not be attributed to a specific amino acid residue), by removing peptides that could not be retraced unambiguously to one specific protein, and by applying a strict threshold on the peptide identification scores. For the human, fly, Arabidopsis, and zebrafish datasets we used a Mascot peptide score threshold of 35; for the mouse dataset we used an Ascore threshold of 19; and from the yeast dataset we took only phosphorylation sites with e-values of 1 × e-04 or lower. For the additional fly dataset we used an dCn threshold of 0.1 and a PeptideProphet threshold of 0.9. Data handling was done with ad hoc Python scripts.
Homologous phosphosites were identified by doing an all-against-all similarity search using the Paralign implementation of the Smith-Waterman algorithm  of all of the full-length proteins for which one or more phosphopeptides were present in the datasets, followed by the identification of high-scoring segment pairs with an e-value of 1 × e-10 or lower in which both the query and the target had the same type of phosphosites at exactly the same position in the alignment (a phosphorylated serine residue should be aligned with a phosphorylated serine residue). Because this procedure does not include any (reciprocal) best hit criteria, all we conclude is that similar sites are homologous; the exact nature of this relationship (orthologous, paralogous) remains unclear. We used a strict e-value threshold of 1 × e-10 for the identification of homologous sequences. The use of a more liberal threshold would increase the overlap (we are now probably missing some homologous phosphorylation events because we did not consider the surrounding sequence to be sufficiently conserved) but would also introduce more noise into an already noisy dataset. In addition, a strict cutoff means that we do not erroneously assume convergently evolved small linear motifs to be homologous (motifs involved in recognition of phosphosites by their kinases tend to be extremely short ).
Expected overlap between datasets assuming independence
The probability that a phosphorylation event in a query dataset is conserved in a target dataset is given by Equation 1.
P(q ∈ OQ, T) = NQ, T/NQ
Where Q is the query dataset, q is a phosphorylation event in Q, T is the target dataset, ∈ means 'element of', ∉ means 'not an element of', OQ, T is the overlap of Q and T (events from Q with a homologous event in T), NQ, T is the number of events in OQ, T, and NQ is the total number of events in Q.
The probability that q has homologs in x of the target datasets is the sum of all possible combinations of presence and absence in all of the target datasets, given x. As an example, we consider target datasets A, B, and C. The probability P that q has homologs in two out of these three datasets is given by Equation 2.
P(q|x = 2) = P(q ∈ OQ, A ⋂ q ∈ OQ, B ⋂ q ∉ OQ, C)+ P(q ∈ OQ, A ⋂ q ∉ OQ, B ⋂ q ∈ OQ, C) + P(q ∉ OQ, A ⋂ q ∈ OQ, B ⋂ q ∈ OQ, C)
Where P(q|x = 2) is the probability that q has homologs in two target datasets, and ⋂ is the 'and' operator.
The expected number of phosphorylation events from a query dataset with homologs in x target datasets is now given by Equation 3.
E (x = i) = P(q|x = i).NQ
In which E is the expected value, and i is a number lower than the total number of datasets.
Relative overlap was calculated by dividing the number of conserved phosphorylation events of the query and target datasets by the number of sites in the query dataset with one or more homologous positions in the target dataset. We identified homologous positions using the results of the all-against-all similarity search described above; a site has a homologous position in a target dataset when the site is part of one or more high-scoring segment pairs in that dataset, irrespective of the specific residue type the site is aligned with.
We identified known domains in the full-length sequence of all proteins with one or more phosphorylation events. Domains were identified with HMMER , using models provided by version 23 of the PFAM database . The location of phosphorylation events relative to these domains was determined using python scripts.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 provides the number of conserved phosphosites per query phosphosite with one more homologous sites in the target dataset.
This work was supported by BioRange project SP 2.3.1 of the Netherlands Bioinformatics Centre (NBIC) and by the Netherlands Proteomics Centre. We thank S Mohammed, M Pinkse, and S Lemeer for their phosphorylation data and valuable comments.
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