Global Quantitative Proteomics Studies Revealed Tissue-Preferential Expression and Phosphorylation of Regulatory Proteins in Arabidopsis.

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1-9 flowers, stage 10-12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48, showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.

Proteomic analysis of lysine acetylation provides strong evidence for involvement of acetylated proteins in plant meiosis and tapetum function.

Protein lysine acetylation (KAC) is a dynamic and reversible post-translational modification that has important biological roles in many organisms. Although KAC has been shown to affect reproductive development and meiosis in yeast and animals, similar studies are largely lacking in flowering plants, especially proteome-scale investigations for particular reproductive stages. Here, we report results from a proteomic investigation to detect the KAC status of the developing rice anthers near the time of meiosis (RAM), providing strong biochemical evidence for roles of many KAC-affected proteins during anther development and meiosis in rice. We identified a total of 1354 KAC sites in 676 proteins. Among these, 421 acetylated proteins with 629 KAC sites are novel, greatly enriching our knowledge on KAC in flowering plants. Gene Ontology enrichment analysis showed chromatin silencing, protein folding, fatty acid biosynthetic process and response to stress to be over-represented. In addition, certain potentially specific KAC motifs in RAM were detected. Importantly, 357 rice meiocyte proteins were acetylated; and four proteins genetically identified to be important for rice tapetum and pollen development were acetylated on 14 KAC sites in total. Furthermore, 47 putative secretory proteins were detected to exhibit acetylated status in RAM. Moreover, by comparing our lysine acetylome with the RAM phosphoproteome we obtained previously, we proposed a correlation between KAC and phosphorylation as a potential modulatory mechanism in rice RAM. This study provides the first global survey of KAC in plant reproductive development, making a promising starting point for further functional analysis of KAC during rice anther development and meiosis.

An Approach to Incorporate Multi-Enzyme Digestion into C-TAILS for C-Terminomics Studies.

Protein C-termini study is still a challenging task and far behind its counterpart, N-termini study. MS based C-terminomics study is often hampered by the low ionization efficiency of C-terminal peptides and the lack of efficient enrichment methods. We previously optimized the C-terminal amine-based isotope labeling of substrates (C-TAILS) method and identified 369 genuine protein C-termini in Escherichia coli. A key limitation of C-TAILS is that the prior protection of amines and carboxylic groups at protein level makes Arg-C as the only specific enzyme in practice. Herein, we report an approach combining multi-enzyme digestion and C-TAILS, which significantly increases the identification rate of C-terminal peptides and consequently improves the applicability of C-TAILS in biological studies. We carry out a systematic study and confirm that the omission of the prior amine protection at protein level has a negligible influence and allows the application of multi-enzyme digestion. We successfully apply five different enzyme digestions to C-TAILS, including trypsin, Arg-C, Lys-C, Lys-N, and Lysarginase. As a result, we identify a total of 722 protein C-termini in E. coli, which is at least 66% more than the results using any single enzyme. Moreover, the favored enzyme and enzyme combination are discovered. Data are available via ProteomeXchange with identifier PXD004275.

An optimized guanidination method for large-scale proteomic studies.

As an ε-amine specific derivatization method, guanidination is widely used in proteomic studies for mainly two reasons: the significant improvement in ionization efficiency and the selective protection of ε-amine. Herein, we employed a systematic comparison of two widely used guanidination approaches and revealed the advantages and disadvantages of each method. The sodium buffer based approach resulted in an unexpected side modification, +57 Da, which is reported for the first time; whereas the ammonium buffer based approach resulted in relatively lower yield. We carried out an optimization study by testing different buffer compositions, pH, temperatures and reaction times, and consequently discovered the optimized guanidination condition. Furthermore, we decoded the +57 Da side product as the addition of C2 H3 NO and proposed a possible mechanism of the side reaction. Importantly, our study demonstrated that mass spectrometry is a powerful tool in discovering minor side reactions which are often impossible by other techniques, and hence suggested that chemical derivatization methods should be investigated more carefully prior to extensive applications in proteomics field.

Proteomic and phosphoproteomic analyses reveal extensive phosphorylation of regulatory proteins in developing rice anthers.

Anther development, particularly around the time of meiosis, is extremely crucial for plant sexual reproduction. Meanwhile, cell-to-cell communication between somatic (especial tapetum) cells and meiocytes are important for both somatic anther development and meiosis. To investigate possible molecular mechanisms modulating protein activities during anther development, we applied high-resolution mass spectrometry-based proteomic and phosphoproteomic analyses for developing rice (Oryza sativa) anthers around the time of meiosis (RAM). In total, we identified 4984 proteins and 3203 phosphoproteins with 8973 unique phosphorylation sites (p-sites). Among those detected here, 1544 phosphoproteins are currently absent in the Plant Protein Phosphorylation DataBase (P3 DB), substantially enriching plant phosphorylation information. Mapman enrichment analysis showed that 'DNA repair','transcription regulation' and 'signaling' related proteins were overrepresented in the phosphorylated proteins. Ten genetically identified rice meiotic proteins were detected to be phosphorylated at a total of 25 p-sites; moreover more than 400 meiotically expressed proteins were revealed to be phosphorylated and their phosphorylation sites were precisely assigned. 163 putative secretory proteins, possibly functioning in cell-to-cell communication, are also phosphorylated. Furthermore, we showed that DNA synthesis, RNA splicing and RNA-directed DNA methylation pathways are extensively affected by phosphorylation. In addition, our data support 46 kinase-substrate pairs predicted by the rice Kinase-Protein Interaction Map, with SnRK1 substrates highly enriched. Taken together, our data revealed extensive protein phosphorylation during anther development, suggesting an important post-translational modification affecting protein activity.

Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development.

SWATH enables precise label-free quantification on proteome scale.

MS-based proteomics has emerged as a powerful tool in biological studies. The shotgun proteomics strategy, in which proteolytic peptides are analyzed in data-dependent mode, enables a detection of the most comprehensive proteome (>10 000 proteins from whole-cell lysate). The quantitative proteomics uses stable isotopes or label-free method to measure relative protein abundance. The isotope labeling strategies are more precise and accurate compared to label-free methods, but labeling procedures are complicated and expensive, and the sample number and types are also limited. Sequential window acquisition of all theoretical mass spectra (SWATH) is a recently developed technique, in which data-independent acquisition is coupled with peptide spectral library match. In principle SWATH method is able to do label-free quantification in an MRM-like manner, which has higher quantification accuracy and precision. Previous data have demonstrated that SWATH can be used to quantify less complex systems, such as spiked-in peptide mixture or protein complex. Our study first time assessed the quantification performance of SWATH method on proteome scale using a complex mouse-cell lysate sample. In total 3600 proteins got identified and quantified without sample prefractionation. The SWATH method shows outstanding quantification precision, whereas the quantification accuracy becomes less perfect when protein abundances differ greatly. However, this inaccuracy does not prevent discovering biological correlates, because the measured signal intensities had linear relationship to the sample loading amounts; thus the SWATH method can predict precisely the significance of a protein. Our results prove that SWATH can provide precise label-free quantification on proteome scale.

Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.

It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 µg each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409.

Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments.

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase action.

Composition and structure of photosystem I in the moss Physcomitrella patens.

Recently, bryophytes, which diverged from the ancestor of seed plants more than 400 million years ago, came into focus in photosynthesis research as they can provide valuable insights into the evolution of photosynthetic complexes during the adaptation to terrestrial life. This study isolated intact photosystem I (PSI) with its associated light-harvesting complex (LHCI) from the moss Physcomitrella patens and characterized its structure, polypeptide composition, and light-harvesting function using electron microscopy, mass spectrometry, biochemical, and physiological methods. It became evident that Physcomitrella possesses a strikingly high number of isoforms for the different PSI core subunits as well as LHCI proteins. It was demonstrated that all these different subunit isoforms are expressed at the protein level and are incorporated into functional PSI-LHCI complexes. Furthermore, in contrast to previous reports, it was demonstrated that Physcomitrella assembles a light-harvesting complex consisting of four light-harvesting proteins forming a higher-plant-like PSI superstructure.

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

A proteome-scale study on in vivo protein Nα-acetylation using an optimized method.

Protein N-terminal acetylation (N(α) -acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α) -modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to be determined prior to the analysis since Lys-containing N(α) -modified peptides may be lost. To address this problem, we hereby present an optimized method by the introduction of double incubation at pH 6.0. We demonstrate with the optimized method that the N(α) -modified peptides can be enriched regardless of whether ε-NH(2) is present or not, and the sample to resin ratio optimization is no longer necessary. Another improvement was accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to alleviate the shortcoming of the reduced charge state of N(α) -modified peptides. We employed a duplicate experiment using 80 µg samples each and identified 922 IPI annotated and 103 IPI unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini data set acquired from a tryptic digest. Furthermore, the reproducibility of the N(α) -acetyl proteome approach was evaluated and its complementarity to the regular proteome approach was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing peptides via the imidazole group was discovered.

Phosphoproteome analysis of Streptomyces development reveals extensive protein phosphorylation accompanying bacterial differentiation.

Streptomycetes are bacterial species that undergo a complex developmental cycle that includes programmed cell death (PCD) events and sporulation. They are widely used in biotechnology because they produce most clinically relevant secondary metabolites. Although Streptomyces coelicolor is one of the bacteria encoding the largest number of eukaryotic type kinases, the biological role of protein phosphorylation in this bacterium has not been extensively studied before. In this issue, the variations of the phosphoproteome of S. coelicolor were characterized. Most distinct Ser/Thr/Tyr phosphorylation events were detected during the presporulation and sporulation stages (80%). Most of these phosphorylations were not reported before in Streptomyces, and included sporulation factors, transcriptional regulators, protein kinases and other regulatory proteins. Several of the identified phosphorylated proteins, FtsZ, DivIVA, and FtsH2, were previously demonstrated to be involved in the sporulation process. We thus established for the first time the widespread occurrence and dynamic features of Ser/Thr/Tyr protein phosphorylation in a bacteria species and also revealed a previously unrecognized phosphorylation motif "x(pT)xEx".

Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples.

Immobilized metal ion affinity chromatography (IMAC) is widely used for phosphopeptide enrichment. However, the robustness, efficiency, and specificity of this technique in large-scale phosphoproteomics studies are still disputed. In this study, we first compared three widely used IMAC materials under three different conditions. Fe(III)-nitrilotriacetic acid (NTA) IMAC resin was chosen due to its superior performance in all tests. We further investigated the solution ionization efficiency change of the phosphoryl group and carboxylic group in different acetonitrile-water solutions and observed that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol was established using Fe(III)-NTA IMAC resin and it proved to be highly selective in the phosphopeptide enrichment of a highly diluted standard sample (1:1000) prior to MALDI MS analysis. We also observed that a higher iron purity led to an increased IMAC enrichment efficiency. The optimized method was then adapted to phosphoproteome analyses of cell lysates of high protein complexity. From either 20 microg of mouse sample or 50 microg of Drosophila melanogaster sample, more than 1000 phosphorylation sites were identified in each study using IMAC-IMAC and LC-MS/MS. We demonstrate efficient separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects.

Author Correction: Cell-type-dependent histone demethylase specificity promotes meiotic chromosome condensation in Arabidopsis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cell-type-dependent histone demethylase specificity promotes meiotic chromosome condensation in Arabidopsis.

Histone demethylation is crucial for proper chromatin structure and to ensure normal development, and requires the large family of Jumonji C (JmjC)-containing demethylases; however, the molecular mechanisms that regulate the substrate specificity of these JmjC-containing demethylases remain largely unknown. Here, we show that the substrate specificity of the Arabidopsis histone demethylase JMJ16 is broadened from Lys 4 of histone H3 (H3K4) alone in somatic cells to both H3K4 and H3K9 when it binds to the meiocyte-specific histone reader MMD1. Consistent with this, the JMJ16 catalytic domain exhibits both H3K4 and H3K9 demethylation activities. Moreover, the JMJ16 C-terminal FYR domain interacts with the JMJ16 catalytic domain and probably restricts its substrate specificity. By contrast, MMD1 can compete with the N-terminal catalytic domain of JMJ16 for binding to the FYR-C domain, thereby expanding the substrate specificity of JMJ16 by preventing the FYR domain from binding to the catalytic domain. We propose that MMD1 and JMJ16 together in male meiocytes promote gene expression in an H3K9me3-dependent manner and thereby contribute to meiotic chromosome condensation.

[Global proteomic and phosphoproteomic analysis of the premature maize anther].

Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.

Activation of necroptosis in multiple sclerosis.

Multiple sclerosis (MS), a common neurodegenerative disease of the CNS, is characterized by the loss of oligodendrocytes and demyelination. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine implicated in MS, can activate necroptosis, a necrotic cell death pathway regulated by RIPK1 and RIPK3 under caspase-8-deficient conditions. Here, we demonstrate defective caspase-8 activation, as well as activation of RIPK1, RIPK3, and MLKL, the hallmark mediators of necroptosis, in the cortical lesions of human MS pathological samples. Furthermore, we show that MS pathological samples are characterized by an increased insoluble proteome in common with other neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Finally, we show that necroptosis mediates oligodendrocyte degeneration induced by TNF-α and that inhibition of RIPK1 protects against oligodendrocyte cell death in two animal models of MS and in culture. Our findings demonstrate that necroptosis is involved in MS and suggest that targeting RIPK1 may represent a therapeutic strategy for MS.

Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum.

Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized metal affinity chromatography, and TiO(2) were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized with a confidence level of 99% or higher. 41.4% of the identified proteins were allocated to the plasma membrane and about half of the phosphorylation sites have not been reported previously. A bioinformatic screen for 12 consensus sequences identified putative kinases for 642 phosphorylation sites. In summary, the protocol deployed here identified several hundred novel phosphorylation sites of cerebellar proteins. Furthermore, it provides a valuable tool to monitor the plasma membrane proteome from any small brain samples of interest under differing physiological or pathophysiological conditions.