Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures

This study describes the gel‐free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC‐MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 ( http://proteomecentral.proteomexchange.org/dataset/PXD000817 ).     

The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion

We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra‐long reverse phase gradient, and identified using a “high‐high” strategy (high mass accuracy on the parent and fragment ions) with higher‐energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: “sites of polar growth,” “vesicle mediated transport,” and “cytoskeleton organization.” In these same GO categories, kinase‐substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase‐substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An‐Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 ( http://proteomecentral.proteomexchange.org/dataset/PXD000715 ).     

Phosphoproteome analysis of Lotus japonicus seeds

In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase‐specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM‐kinase target motifs, X‐X‐pS/pT‐Q‐X‐X, were detected in cotyledons. Moreover, by real‐time RT‐PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM‐kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 ( http://proteomecentral.proteomexchange.org/dataset/PXD000053 ).     

Phosphoproteomic analysis of chromoplasts from sweet orange during fruit ripening

Like other types of plastids, chromoplasts have essential biosynthetic and metabolic activities which may be regulated via post‐translational modifications, such as phosphorylation, of their resident proteins. We here report a proteome‐wide mapping of in vivo phosphorylation sites in chromoplast‐enriched samples prepared from sweet orange [Citrus sinensis (L.) Osbeck] at different ripening stages by titanium dioxide‐based affinity chromatography for phosphoprotein enrichment with LC‐MS/MS. A total of 109 plastid‐localized phosphoprotein candidates were identified that correspond to 179 unique phosphorylation sites in 135 phosphopeptides. On the basis of Motif‐X analysis, two distinct types of phosphorylation sites, one as proline‐directed phosphorylation motif and the other as casein kinase II motif, can be generalized from these identified phosphopeptides. While most identified phosphoproteins show high homology to those already identified in plastids, approximately 22% of them are novel based on BLAST search using the public databases PhosPhAt and P³DB. A close comparative analysis showed that approximately 50% of the phosphoproteins identified in citrus chromoplasts find obvious counterparts in the chloroplast phosphoproteome, suggesting a rather high‐level of conservation in basic metabolic activities in these two types of plastids. Not surprisingly, the phosphoproteome of citrus chromoplasts is also characterized by the lack of phosphoproteins involved in photosynthesis and by the presence of more phosphoproteins implicated in stress/redox responses. This study presents the first comprehensive phosphoproteomic analysis of chromoplasts and may help to understand how phosphorylation regulates differentiation of citrus chromoplasts during fruit ripening.     

The Protein Phosphorylation Landscape of Mouse Spermatids during Spermiogenesis

The characteristic tadpole shape of sperm is formed from round spermatids via spermiogenesis, a process which results in dramatic morphological changes in the final stage of spermatogenesis in the testis. Protein phosphorylation, as one of the most important post‐translational modifications, can regulate spermiogenesis; however, the phosphorylation events taking place during this process have not been systematically analyzed. In order to better understand the role of phosphorylation in spermiogenesis, large‐scale phosphoproteome profiling is performed using IMAC and TiO₂ enrichment. In total, 13 835 phosphorylation sites, in 4196 phosphoproteins, are identified in purified mouse spermatids undergoing spermiogenesis in two biological replicates. Overall, 735 testis‐specific proteins are identified to be phosphorylated, and are expressed at high levels during spermiogenesis. Gene ontology analysis shows enrichment of the identified phosphoproteins in terms of histone modification, cilium organization, centrosome and the adherens junction. Further characterization of the kinase‐substrate phosphorylation network demonstrates enrichment of phosphorylation substrates related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis shows wide phosphoregulation across a diverse range of processes during spermiogenesis and can help to further characterize the process of sperm generation. All MS data are available via ProteomeXchange with the identifier PXD011890.     

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 ( http://proteomecentral.proteomexchange.org/dataset/PXD001684 ). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.     

Global analysis of phosphoproteome dynamics in embryonic development of zebrafish (Danio rerio)

The zebrafish (Danio rerio) is a popular animal model used for studies on vertebrate development and organogenesis. Recent research has shown a similarity of approximately 70% between the human and zebrafish genomes and about 84% of human disease‐causing genes have common ancestry with that of the zebrafish genes. Zebrafish embryos have a number of desirable features, including transparency, a large size, and rapid embryogenesis. Protein phosphorylation is a well‐known PTM, which can carry out various biological functions. Recent MS developments have enabled the study of global phosphorylation patterns by using MS‐based proteomics coupled with titanium dioxide phosphopeptide enrichment. In the present study, we identified 3500 nonredundant phosphorylation sites on 2166 phosphoproteins and quantified 1564 phosphoproteins in developing embryos of zebrafish. The distribution of Ser/Thr/Tyr phosphorylation sites in zebrafish embryos was found to be 88.9, 10.2, and 0.9%, respectively. A potential kinase motif was predicted using Motif‐X analysis, for 80% of the identified phosphorylation sites, with the proline‐directed motif appearing most frequently, and 35 phosphorylation sites having the pSF motif. In addition, we created six phosphoprotein clusters based on their dynamic pattern during the development of zebrafish embryos. Here, we report the largest dataset of phosphoproteins in zebrafish embryos and our results can be used for further studies on phosphorylation sites or phosphoprotein dynamics in zebrafish embryos.     

Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America and has become a public health concern in the United States and areas of Europe. The possibility that kinase inhibitors represent novel anti‐parasitic agents is currently being explored. However, fundamental understanding of the cell‐signaling networks requires the detailed analysis of the involved phosphorylated proteins. Here, we have performed a comprehensive MS‐based phosphorylation mapping of phosphoproteins from T. cruzi epimastigote forms. Our LC‐MS/MS, dual‐stage fragmentation, and multistage activation analysis has identified 237 phosphopeptides from 119 distinct proteins. Furthermore, 220 phosphorylation sites were unambiguously mapped: 148 on serine, 57 on threonine, and 8 on tyrosine. In addition, immunoprecipitation and Western blotting analysis confirmed the presence of at least seven tyrosine‐phosphorylated proteins in T. cruzi. The identified phosphoproteins were subjected to Gene Ontology, InterPro, and BLAST analysis, and categorized based on their role in cell structure, motility, transportation, metabolism, pathogenesis, DNA/RNA/protein turnover, and signaling. Taken together, our phosphoproteomic data provide new insights into the molecular mechanisms governed by protein kinases and phosphatases in T. cruzi. We discuss the potential roles of the identified phosphoproteins in parasite physiology and drug development.     

Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms

The RAS‐RAF‐MEK‐ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large‐scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 ( http://proteomecentral.proteomexchange.org/dataset/PXD003908 ).     

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non‐pathogenic Pseudomonas species

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.     

A survey of the Arabidopsis thaliana mitochondrial phosphoproteome

Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2‐DE separations of Arabidopsis mitochondrial proteins and affinity‐enriched phosphoproteins using the Pro‐Q Diamond phospho‐specific in‐gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.     

A Rapid Screening Assay to Search for Phosphorylated Proteins in Tissue Extracts

Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO₂ enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.     

Phosphoproteome profile of human lung cancer cell line A549

As an in vitro model for type II human lung cancer, A549 cells resist cytotoxicity via phosphorylation of proteins as demonstrated by many studies. However, to date, no large-scale phosphoproteome investigation has been conducted on A549. Here, we performed a systematical analysis of the phosphoproteome of A549 by using mass spectrometry (MS)-based strategies. This investigation led to the identification of 337 phosphorylation sites on 181 phosphoproteins. Among them, 67 phosphoproteins and 230 phosphorylation sites identified appeared to be novel with no previous characterization in lung cancer. Based on their known functions as reported in the literature, these phosphoproteins were functionally organized into highly interconnected networks. Western blotting and immunohistochemistry analyses were performed to validate the expression of a bottleneck phosphoprotein YAP1 in cancer cell lines and tissues. This dataset provides a valuable resource for further studies on phosphorylation and lung carcinogenesis.     

Construction of human activity‐based phosphorylation networks

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.     

A peptide resource for the analysis of Staphylococcus aureus in host–pathogen interaction studies

Staphylococcus aureus is an opportunistic human pathogen, which can cause life‐threatening disease. Proteome analyses of the bacterium can provide new insights into its pathophysiology and important facets of metabolic adaptation and, thus, aid the recognition of targets for intervention. However, the value of such proteome studies increases with their comprehensiveness. We present an MS–driven, proteome‐wide characterization of the strain S. aureus HG001. Combining 144 high precision proteomic data sets, we identified 19 109 peptides from 2088 distinct S. aureus HG001 proteins, which account for 72% of the predicted ORFs. Peptides were further characterized concerning pI, GRAVY, and detectability scores in order to understand the low peptide coverage of 8.7% (19 109 out of 220 245 theoretical peptides). The high quality peptide‐centric spectra have been organized into a comprehensive peptide fragmentation library (SpectraST) and used for identification of S. aureus‐typic peptides in highly complex host–pathogen interaction experiments, which significantly improved the number of identified S. aureus proteins compared to a MASCOT search. This effort now allows the elucidation of crucial pathophysiological questions in S. aureus‐specific host–pathogen interaction studies through comprehensive proteome analysis. The S. aureus‐specific spectra resource developed here also represents an important spectral repository for SRM or for data‐independent acquisition MS approaches. All MS data have been deposited in the ProteomeXchange with identifier PXD000702 ( http://proteomecentral.proteomexchange.org/dataset/PXD000702 ).     

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.     

In‐depth characterization of the tomato fruit pericarp proteome

Since the genome of Solanum lycopersicum L. was published in 2012, some studies have explored its proteome although with a limited depth. In this work, we present an extended characterization of the proteome of the tomato pericarp at its ripe red stage. Fractionation of tryptic peptides generated from pericarp proteins by off‐line high‐pH reverse‐phase phase chromatography in combination with LC‐MS/MS analysis on a Fisher Scientific Q Exactive and a Sciex Triple‐TOF 6600 resulted in the identification of 8588 proteins with a 1% FDR both at the peptide and protein levels. Proteins were mapped through GO and KEGG databases and a large number of the identified proteins were associated with cytoplasmic organelles and metabolic pathways categories. These results constitute one of the most extensive proteome datasets of tomato so far and provide an experimental confirmation of the existence of a high number of theoretically predicted proteins. All MS data are available in the ProteomeXchange repository with the dataset identifiers PXD004947 and PXD004932.