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 ).     

Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin

Studies of the secretomes of microbes grown on insoluble substrates are important for the discovery of novel proteins involved in biomass conversion. However, data in literature and this study indicate that secretome samples tend to be contaminated with cytoplasmic proteins. We have examined the secretome of the Gram‐negative soil bacterium Cellvibrio japonicus using a simple plate‐based culturing technique that yields samples with high fractions (60–75%) of proteins that are predicted to be secreted. By combining this approach with label‐free quantification using the MaxLFQ algorithm, we have mapped and quantified proteins secreted by C. japonicus during growth on α‐ and β‐chitin. Hierarchical clustering of the detected protein quantities revealed groups of up‐regulated proteins that include all five putative C. japonicus chitinases as well as a chitin‐specific lytic polysaccharide monooxygenase (CjLPMO10A). A small set of secreted proteins were co‐regulated with known chitin‐specific enzymes, including several with unknown catalytic functions. These proteins provide interesting targets for further studies aimed at unraveling the enzymatic machineries used by C. japonicus for recalcitrant polysaccharide degradation. Studies of chitin degradation indicated that C. japonicus indeed produces an efficient chitinolytic enzyme cocktail. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD002843 ( http://proteomecentral.proteomexchange.org/dataset/PXD002843 ).     

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 ).     

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.     

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.     

Qualitative and quantitative analysis of the adult Drosophila melanogaster proteome

Drosophila melanogaster is one of the most widely used model organisms in life sciences. Mapping its proteome is of great significance for understanding the biological characteristics and tissue functions of this species. However, the comprehensive coverage of its proteome remains a challenge. Here, we describe a high‐coverage analysis of whole fly through a 1D gel electrophoresis and LC‐MS/MS approach. By combining the datasets of two types of SDS‐PAGE and two kinds of tagmata, the high‐coverage analysis resulted in the identification of 5262 genes, which correspond to 38.23% of the entire coding genes. Moreover, we found that the fly head and body have different molecular weight distributions of their proteomes when the proteins were resolved with SDS‐PAGE and image analysis of the stained gel. This phenomenon was further confirmed by both label‐free and isobaric tags for relative and absolute quantitation‐based quantitative approaches. The consistent results of the two different quantitation methods also demonstrated the stability and accuracy of the LC‐MS/MS platform. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD000454 and PXD000455 ( http://proteomecentral.proteomexchange.org/dataset/PXD000454 ; ( http://proteomecentral.proteomexchange.org/dataset/PXD000455 ).     

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.     

一种鉴定金花茶人工杂交F_1代杂种苗的简易方法

本试验选用了两个以金花茶作父本的人工杂交组合F_1代实生苗,经根尖体细胞染色体观察鉴定四倍体杂种。一、云南野山茶(Camellia pitardii var.yunnanica六倍体)×金花茶(C.Chrysantha二倍体)。结果为:所获56株子叶和下胚轴为红色的F_1代杂种苗中,有55株(98.2％)是真正的四倍体杂种(X~2=0.0128,D.f.=1,0.95>p>0.90);所获50株子叶和下胚轴为黄白色的杂种苗中,有13株(26％)为真正四倍体杂种(X~2=27.38,D.f.=13,0.02>p>0.01)。二、云南山茶花(C.reticulata六倍体)×金花茶。结果为:在78株子叶和下胚轴为红色的杂交苗中,有77株(98.7％)为真正四倍体杂种(X~2=0.0128,D.f.=1,0.95>p>0.90);而在78株子叶及下胚轴为黄白色的杂种苗中,只有21株(26.9％)是真正四倍体杂种(X~2=41.65,D.f.=24,0.02>p>0.01) 在多数杂种实生苗中的这种红色素,是因种子直感现象而发生的父本金花茶的一种遗传性状。金花茶的另一特征——多子叶现象(3枚以上),则在一些F_1代杂种苗或杂种种子中表现得不明显。因此,利用F_1代杂种苗子叶和下胚轴所具有的红色特征,在杂种种子萌发期用来鉴别以金花茶为亲本的F_1代杂种的真伪,是一种简便、快速的、有发展前途的方法。     

Secondary somatic embryogenesis and plant regeneration in myrtle (Myrtus communis L.)

Immature seeds, as well as hypocotyls and cotyledons excised from seedlings of Myrtus communis L., were cultured on media containing half-strength Murashige and Skoog macronutrients (MS/2) with combinations of auxins and cytokinins, in order to study their morphogenetic competence. Somatic embryogenesis was obtained from cotyledons, hypocotyls and 2-month-old immature seeds with 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The percentage of explants showing this primary somatic embryogenesis ranged from 4% for hypocotyls to 12% for 2-month-old immature seeds. In the latter, somatic embryogenesis was also obtained in media containing 2,4-D plus a cytokinin, and with only a cytokinin. Somatic embryos obtained from hypocotyls, cotyledons or immature seeds were able to develop on MS/2 medium without plant growth regulators. Subculture of primary somatic embryos obtained from immature seeds on MS/2 medium without plant growth regulators gave rise to clusters with secondary somatic embryos and embryogenic calli. These clusters were subcultured every 8 weeks, and they were the source of highly embryogenic cultures. An average of 10% of the secondary somatic embryos developed into plantlets in each subculture. Therefore, the same culture on MS/2 medium without growth regulators yielded both plantlets and de novo secondary embryos. Received: 6 April 1998 / Revision received: 10 July 1998 / Accepted: 21 July 1998     

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 ).     

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 primary human multiple myeloma cells

Multiple myeloma (MM) is a malignant disorder of differentiated B cells. Clonal expansion of the tumor results in the excessive production of monoclonal immunoglobulin (Ig) which is a diagnostic feature of this disease. Previous investigations have demonstrated the alteration of the ERK, jun kinase, STAT, and AKT kinase signaling cascades in MM cells, suggesting that deregulated phosphorylation may contribute to MM pathogenesis. However, systematic analysis of the phosphoproteome in MM cells has not been reported. Here, we described a large-scale phosphorylation analysis of primary MM cells. Using a separation strategy involving immunomagnetic bead-positive selection of MM cells, preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and titanium dioxide 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 primary MM cells. This analysis led to the identification of 530 phosphorylation sites from 325 unique phosphopeptides corresponding to 260 proteins at false positive rate (FPR) of 1.3%. This dataset provides an important resource for future studies on phosphorylation and carcinogenesis analysis of multiple myeloma.     

The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Calibration plot for proteomics: A graphical tool to visually check the assumptions underlying FDR control in quantitative experiments

In MS‐based quantitative proteomics, the FDR control (i.e. the limitation of the number of proteins that are wrongly claimed as differentially abundant between several conditions) is a major postanalysis step. It is classically achieved thanks to a specific statistical procedure that computes the adjusted p‐values of the putative differentially abundant proteins. Unfortunately, such adjustment is conservative only if the p‐values are well‐calibrated; the false discovery control being spuriously underestimated otherwise. However, well‐calibration is a property that can be violated in some practical cases. To overcome this limitation, we propose a graphical method to straightforwardly and visually assess the p‐value well‐calibration, as well as the R codes to embed it in any pipeline. All MS data have been deposited in the ProteomeXchange with identifier PXD002370 ( http://proteomecentral.proteomexchange.org/dataset/PXD002370 ).     

Large-scale analysis of phosphorylated proteins in maize leaf

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.     

Proteome profile of the endomembrane of developing coleoptiles from switchgrass (Panicum virgatum)

The cost‐effective production of biofuels from lignocellulosic material will likely require manipulation of plant biomass, specifically cell walls. The North American native prairie grass Panicum virgatum (switchgrass) is seen as a potential biofuel crop with an array of genetic resources currently being developed. We have characterized the endomembrane proteome of switchgrass coleoptiles to provide additional information to the switchgrass community. In total, we identified 1750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD001351 ( http://proteomecentral.proteomexchange.org/dataset/PXD001351 ).