Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO(2) -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.     

In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach

In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast‐enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO₂) affinity chromatography and were subsequently analysed using LC‐ESI‐MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na⁺/H⁺ antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.     

Phosphoproteomic identification and phylogenetic analysis of ribosomal P-proteins in Populus dormant terminal buds

To better understand the role that reversible phosphorylation plays in woody plant ribosomal P-protein function, we initiated a phosphoproteomic investigation of P-proteins from Populus dormant terminal buds. Using gel-free (in-solution) protein digestion and phosphopeptide enrichment combined with a nanoUPLC–ESI–MS/MS strategy, we identified six phosphorylation sites on eight P-proteins from Populus dormant terminal buds. Among these, six Ser sites and one Thr site were identified in the highly conserved C-terminal region of eight P-proteins of various P-protein subfamilies, including two P0, two P1, three P2 and one P3 protein. Among these, the Thr site was shown to be novel and has not been identified in any other organisms. Sequence analysis indicated that the phosphothreonine sites identified in the C-terminus of Ptr RPP2A exclusively occurred in woody species of Populus, etc. The identified phosphopeptides shared a common phosphorylation motif of (S/T)XX(D/E) and may be phosphorylated in vivo by casein kinase 2 as suggested by using Scansite analysis. Furthermore, phylogenetic analysis suggested that divergence of P2 also occurred in Populus, including type I and type II. To the best of our knowledge, this is the first systematic phosphoproteomic and phylogenetic analysis of P-proteins in woody plants, the results of which will provide a wealth of resources for future understanding and unraveling of the regulatory mechanisms of Populus P-protein phosphorylation during the maintenance of dormancy.     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.     

Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA metabolism

Most regulatory pathways are governed by the reversible phosphorylation of proteins. Recent developments in mass spectrometry-based technology allow the large-scale analysis of protein phosphorylation. Here, we show the application of immobilized metal affinity chromatography to purify phosphopeptides from Arabidopsis extracts. Phosphopeptide sequences were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS/MS). A total of 79 unique phosphorylation sites were determined in 22 phosphoproteins with a putative role in RNA metabolism, including splicing of mRNAs. Among these phosphoproteins, 12 Ser/Arg-rich (SR) splicing factors were identified. A conserved phosphorylation site was found in most of the phosphoproteins, including the SR proteins, suggesting that these proteins are targeted by the same or a highly related protein kinase. To test this hypothesis, Arabidopsis SR protein-specific kinase 4 (SRPK4) that was initially identified as an interactor of SR proteins was tested for its ability to phosphorylate the SR protein RSp31. In vitro kinase assays showed that all in vivo phosphorylation sites of RSp31 were targeted by SRPK4. These data suggest that the plant mRNA splicing machinery is a major target of phosphorylation and that a considerable number of proteins involved in RNA metabolism may be targeted by SRPKs.     

Quantitative phosphoproteomics strategies for understanding protein kinase-mediated signal transduction pathways

Protein phosphorylation is a central regulatory mechanism of cell signaling pathways. This highly controlled biochemical process is involved in most cellular functions, and defects in protein kinases and phosphatases have been implicated in many diseases, highlighting the importance of understanding phosphorylation-mediated signaling networks. However, phosphorylation is a transient modification, and phosphorylated proteins are often less abundant. Therefore, the large-scale identification and quantification of phosphoproteins and their phosphorylation sites under different conditions are one of the most interesting and challenging tasks in the field of proteomics. Both 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry serve as key phosphoproteomic technologies in combination with prefractionation, such as enrichment of phosphorylated proteins/peptides. Recently, new possibilities for quantitative phosphoproteomic analysis have been offered by technical advances in sample preparation, enrichment, separation, instrumentation, quantification and informatics. In this article, we present an overview of several strategies for quantitative phosphoproteomics and discuss how phosphoproteomic analysis can help to elucidate signaling pathways that regulate various cellular processes.     

Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry

The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.     

Unraveling molecular complexity of phosphorylated human cardiac troponin I by top down electron capture dissociation/electron transfer dissociation mass spectrometry

Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI approximately 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser(22) and a novel site at Ser(76)/Thr(77), each with partial occupancy (Ser(22): 53%; Ser(76)/Thr(77): 36%). Top down MS(3) analysis of diphosphorylated cTnI revealed occupancy of Ser(23) only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser(22/23) pair. Top down MS of cTnI provides unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing establishment of the relevance of such modifications to physiological functions and disease status.     

Comprehensive phosphoproteome analysis of INS-1 pancreatic beta-cells using various digestion strategies coupled with liquid chromatography-tandem mass spectrometry

Type 2 diabetes results from aberrant regulation of the phosphorylation cascade in beta-cells. Phosphorylation in pancreatic beta-cells has not been examined extensively, except with regard to subcellular phosphoproteomes using mitochondria. Thus, robust, comprehensive analytical strategies are needed to characterize the many phosphorylated proteins that exist, because of their low abundance, the low stoichiometry of phosphorylation, and the dynamic regulation of phosphoproteins. In this study, we attempted to generate data on a large-scale phosphoproteome from the INS-1 rat pancreatic beta-cell line using linear ion trap MS/MS. To profile the phosphoproteome in-depth, we used comprehensive phosphoproteomic strategies, including detergent-based protein extraction (SDS and SDC), differential sample preparation (in-gel, in-solution digestion, and FASP), TiO2 enrichment, and MS replicate analyses (MS2-only and multiple-stage activation). All spectra were processed and validated by stringent multiple filtering using target and decoy databases. We identified 2467 distinct phosphorylation sites on 1419 phosphoproteins using 4 mg of INS-1 cell lysate in 24 LC-MS/MS runs, of which 683 (27.7%) were considered novel phosphorylation sites that have not been characterized in human, mouse, or rat homologues. Our informatics data constitute a rich bioinformatics resource for investigating the function of reversible phosphorylation in pancreatic beta-cells. In particular, novel phosphorylation sites on proteins that mediate the pathology of type 2 diabetes, such as Pdx-1, Nkx.2, and Srebf1, will be valuable targets in ongoing phosphoproteomics studies.     

A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H(+) pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca(2+) pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H(+) pumps and H(+):cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent.     

A Phos‐tag SDS‐PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1

MEK1, an essential component of the mitogen‐activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS‐based phosphoproteomic methods. By using Phos‐tag SDS‐PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos‐tag‐based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time‐course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti‐HaloTag antibody, we analyzed a HaloTag‐fused MEK1 protein and 12 potential phosphorylation‐site‐directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr‐292, Ser‐298, Thr‐386, and Thr‐388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser‐218 and Ser‐222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr‐292, Ser‐298, Thr‐386, and Thr‐388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor‐specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK‐733, PD98059, and U0126.     

Large-scale analysis of protein phosphorylation in Populus leaves

Protein phosphorylation is a key regulatory factor in all aspects of plant biology; most regulatory pathways are governed by the reversible phosphorylation of proteins. To better understand the role that phosphorylated proteins play in a woody model plant, we performed a systemic analysis of the phosphoproteome from Populus leaves using high accuracy NanoLC–MS/MS in combination with biochemical enrichments using strong cation exchange chromatography and titanium dioxide chromatography. We identified 104 phosphopeptides from 94 phosphoproteins and determined 111 phosphorylation sites including 93 occurring on serine residues and 18 on threonine residues. The identified phosphoproteins are involved in a wide variety of metabolic processes. Among these identified phosphoproteins, 68 phosphorylation sites (72 %) were located outside of conserved domains. The identified phosphopeptides share a common phosphorylation motif of pS/pT-P/D-S/A. These data suggest that the Populus metabolism and gene regulation machinery are major targets of phosphorylation. To our knowledge, this is the first gel-free, large-scale phosphoproteomics analysis in woody plants. The identified phosphorylation sites will be a valuable resource for many fields of plant biology, and information gained from the study will provide a better understanding of protein phosphorylation.     

Major Cdk5-dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis

Amphiphysin 1 (amph 1) is an endocytic protein enriched in the nerve terminals that functions in the clathrin-mediated endocytosis. It acts as membrane curvature sensor, a linker of clathrin coat proteins, and an enhancer of dynamin Guanosine Triphosphatase (GTPase) activity. Amph 1 undergoes phosphorylation by cyclin-dependent kinase 5 (Cdk5), at five phosphorylation sites, serine 262, 272, 276, 285, and threonine 310, as determined by mass spectrometry (MS). We show here that Cdk5-dependent phosphorylation of amph 1 is enhanced in the presence of lipid membranes. Analysis by tandem liquid chromatograph MS revealed that the phosphorylation occurs at two phosphorylation sites. The phosphorylation was markedly decreased by mutation either Ser276 or Ser285 of amph 1 to alanine (S276A and S285A). Furthermore, mutation of both sites (S276, 285A) completely eliminated the phosphorylation. Functional studies indicated that binding of amph 1 to lipid membrane was attenuated by Cdk5-dependent phosphorylation of wild type amph 1, but not of the S276, 285A form. Interestingly, endocytosis was increased in rat pheochromocytoma cells expressing amph 1 S276, 285A in comparison with wild type. These results suggest that Ser276 and Ser285 are regulatory Cdk5 phosphorylation sites of amph 1 in the lipid-bound state. Phosphorylation at these sites alters binding of amph 1 to lipid membranes, and may be an important regulatory aspect in the regulation of synaptic vesicle endocytosis.     

The stoichiometry of protein phosphorylation in adipocyte lipid droplets: Analysis by N‐terminal isotope tagging and enzymatic dephosphorylation

Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non‐phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC‐MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a β‐3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone‐sensitive lipase which was 80% phosphorylated at the previously reported site, Ser‐559, and the lipid surface protein perilipin, which was phosphorylated by ～60 and ～40% at previously unreported sites, Ser‐410 and Ser‐460.     

Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/. Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions ("adherens junction," "tight junction," and "focal adhesion") and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.     

Bioinformatic prediction and confirmation of beta-adducin as a novel substrate of glycogen synthase kinase 3

It is important to identify the true substrates of protein kinases because this illuminates the primary function of any kinase. Here, we used bioinformatics and biochemical validation to identify novel brain substrates of the Ser/Thr kinase glycogen synthase kinase 3 (GSK3). Briefly, sequence databases were searched for proteins containing a conserved GSK3 phosphorylation consensus sequence ((S/T)PXX(S/T)P or (S/T)PXXX(S/T)P), as well as other criteria of interest (e.g. brain proteins). Importantly, candidates were highlighted if they had previously been reported to be phosphorylated at these sites by large-scale phosphoproteomic studies. These criteria identified the brain-enriched cytoskeleton-associated protein β-adducin as a likely substrate of GSK3. To confirm this experimentally, it was cloned and subjected to a combination of cell culture and in vitro kinase assays that demonstrated direct phosphorylation by GSK3 in vitro and in cells. Phosphosites were mapped to three separate regions near the C terminus and confirmed using phosphospecific antibodies. Prior priming phosphorylation by Cdk5 enhanced phosphorylation by GSK3. Expression of wild type, but not non-phosphorylatable (GSK3 insensitive), β-adducin increased axon and dendrite elongation in primary cortical neurons. Therefore, phosphorylation of β-adducin by GSK3 promotes efficient neurite outgrowth in neurons.     

Comparative phosphoproteomic analysis of mammalian glomeruli reveals conserved podocin C‐terminal phosphorylation as a determinant of slit diaphragm complex architecture

Glomerular biology is dependent on tightly controlled signal transduction networks that control phosphorylation of signaling proteins such as cytoskeletal regulators or slit diaphragm proteins of kidney podocytes. Cross‐species comparison of phosphorylation events is a powerful mean to functionally prioritize and identify physiologically meaningful phosphorylation sites. Here, we present the result of phosphoproteomic analyses of cow and rat glomeruli to allow cross‐species comparisons. We discovered several phosphorylation sites with potentially high biological relevance, e.g. tyrosine phosphorylation of the cytoskeletal regulator synaptopodin and the slit diaphragm protein neph‐1 (Kirrel). Moreover, cross‐species comparisons revealed conserved phosphorylation of the slit diaphragm protein nephrin on an acidic cluster at the intracellular terminus and conserved podocin phosphorylation on the very carboxyl terminus of the protein. We studied a highly conserved podocin phosphorylation site in greater detail and show that phosphorylation regulates affinity of the interaction with nephrin and CD2AP. Taken together, these results suggest that species comparisons of phosphoproteomic data may reveal regulatory principles in glomerular biology. All MS data have been deposited in the ProteomeXchange with identifier PXD001005 ( http://proteomecentral.proteomexchange.org/dataset/PXD001005 ).     

A probability-based approach for high-throughput protein phosphorylation analysis and site localization

Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.     

Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae

Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.     

A phosphoproteomic landscape of rice (Oryza sativa) tissues

Protein phosphorylation is an important posttranslational modification that regulates various plant developmental processes. Here, we report a comprehensive, quantitative phosphoproteomic profile of six rice tissues, including callus, leaf, root, shoot meristem, young panicle and mature panicle from Nipponbare by employing a mass spectrometry (MS)‐based, label‐free approach. A total of 7171 unique phosphorylation sites in 4792 phosphopeptides from 2657 phosphoproteins were identified, of which 4613 peptides were differentially phosphorylated (DP) among the tissues. Motif‐X analysis revealed eight significantly enriched motifs, such as [sP], [Rxxs] and [tP] from the rice phosphosites. Hierarchical clustering analysis divided the DP peptides into 63 subgroups, which showed divergent spatial‐phosphorylation patterns among tissues. These clustered proteins are functionally related to nutrition uptake in roots, photosynthesis in leaves and tissue differentiation in panicles. Phosphorylations were specific in the tissues where the target proteins execute their functions, suggesting that phosphorylation might be a key mechanism to regulate the protein activity in different tissues. This study greatly expands the rice phosphoproteomic dataset, and also offers insight into the regulatory roles of phosphorylation in tissue development and functions.