A gas phase fractionation acquisition scheme integrating ion mobility for rapid diaPASEF library generation.

Data independent acquisition (DIA/SWATH) MS is a primary strategy in quantitative proteomics. diaPASEF is a recent adaptation using trapped ion mobility spectrometry (TIMS) to improve selectivity/sensitivity. Complex DIA spectra are typically analyzed with reference to spectral libraries. The best-established method for generating libraries uses offline fractionation to increase depth of coverage. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow DIA windows that cover different mass ranges of the complete precursor space, have been introduced that performed comparably to deep offline fractionation-based libraries. We investigated whether an analogous GPF-based approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data. We developed a rapid library generation approach using an IM-GPF acquisition scheme in the m/z versus 1/K0 space requiring seven injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation. We found that library generation by IM-GPF outperformed direct library generation from diaPASEF and had performance approaching that of the deep library. This establishes the IM-GPF scheme as a pragmatic approach to rapid library generation for analysis of diaPASEF data.

Susceptibility of Rice Crop to Salt Threat: Proteomic, Metabolomic, and Physiological Inspections.

Rice is a staple food crop worldwide; however, salinity stress is estimated to reduce its global production by 50%. Knowledge about initial molecular signaling and proteins associated with sensing salinity among crop plants is limited. We characterized early salt effects on the proteome and metabolome of rice tissues. Omics results were validated by western blotting and multiple reaction monitoring assays and integrated with physiological changes. We identified 8160 proteins and 2045 metabolites in rice tissues. Numerous signaling pathways were induced rapidly or partially by salinity. Combined data showed the most susceptible proteins or metabolites in each pathway that likely affected the sensitivity of rice to salinity, such as PLA1, BON3 (involved in sensing stress), SnRK2, pro-resilin, GDT1, G-proteins, calmodulin activators (Ca2+ and abscisic acid signaling), MAPK3/5, MAPKK1/3 (MAPK pathway), SOS1, ABC F/D, PIP2-7, and K+ transporter-23 (transporters), OPR1, JAR1, COL1, ABA2, and MAPKK3 (phytohormones). Additionally, our results expanded the stress-sensing function of receptor-like kinases, phosphatidylinositols, and Na+ sensing proteins (IPUT1). Combined analyses revealed the most sensitive components of signaling pathways causing salt-susceptibility in rice and suggested potential targets for crop improvement.

Tyrosine Phosphorylation Profiling Revealed the Signaling Network Characteristics of CAMKK2 in Gastric Adenocarcinoma.

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine protein kinase which functions via the calcium-triggered signaling cascade with CAMK1, CAMK4, and AMPKα as the immediate downstream substrates. CAMKK2 is reported to be overexpressed in gastric cancer; however, its signaling mechanism is poorly understood. We carried out label-free quantitative tyrosine phosphoproteomics to investigate tyrosine-mediated molecular signaling associated with CAMKK2 in gastric cancer cells. Using a high-resolution Orbitrap Fusion Tribrid Fourier-transform mass spectrometer, we identified 350 phosphotyrosine sites mapping to 157 proteins. We observed significant alterations in 81 phosphopeptides corresponding to 63 proteins upon inhibition of CAMKK2, among which 16 peptides were hyperphosphorylated corresponding to 13 proteins and 65 peptides were hypophosphorylated corresponding to 51 proteins. We report here that the inhibition of CAMKK2 leads to changes in the phosphorylation of several tyrosine kinases such as PKP2, PTK2, EPHA1, EPHA2, PRKCD, MAPK12, among others. Pathway analyses revealed that proteins are differentially phosphorylated in response to CAMKK2 inhibition involved in focal adhesions, actin cytoskeleton, axon guidance, and signaling by VEGF. The western blot analysis upon inhibition and/or silencing of CAMKK2 revealed a decrease in phosphorylation of PTK2 at Y925, c-JUN at S73, and STAT3 at Y705, which was in concordance with the mass spectrometry data. The study indicates that inhibition of CAMKK2 has an anti-oncogenic effect in gastric cells regulating phosphorylation of STAT3 through PTK2/c-JUN in gastric cancer.

The Core Human MicroRNAs Regulated by Toxoplasma gondii.

BACKGROUND: Toxoplasma gondii (T. gondii) is an intracellular zoonotic protozoan parasite known to effectively modulate the host system for its survival. A large number of microRNAs (miRNAs) regulated by different strains of T. gondii in diverse types of host cells/tissues/organs have been reported across multiple studies. OBJECTIVE: We aimed to decipher the complexity of T. gondii regulated spectrum of miRNAs to derive a set of core miRNAs central to different strains of T. gondii infection in diverse human cell lines. METHODS: We first assembled miRNAs hat are regulated by T. gondii altered across the various assortment of infections and time points of T. gondii infection in multiple cell types. For these assembled datasets, we employed specific criteria to filter the core miRNAs regulated by T. gondii. Subsequently, accounting for the spectrum of miRNA-mRNA target combinations, we applied a novel confidence criterion to extract their core experimentally-validated mRNA targets in human cell systems. RESULTS: This analysis resulted in the extraction of 74 core differentially regulated miRNAs and their 319 high-confidence mRNA targets. Based on these core miRNA-mRNA pairs, we derived the central biological processes perturbed by T. gondii in diverse human cell systems. Further, our analysis also resulted in the identification of novel autocrine/paracrine signalling factors that could be associated with host response modulated by T. gondii. CONCLUSION: The current analysis derived a set of core miRNAs, their targets, and associated biological processes fine-tuned by T. gondii for its survival within the invaded cells.

Towards Phytopathogen Diagnostics? Coconut Bud Rot Pathogen Phytophthora palmivora Mycelial Proteome Analysis Informs Genome Annotation.

Planetary agriculture stands to benefit immensely from phytopathogen diagnostics, which would enable early detection of pathogens with harmful effects on crops. For example, Phytophthora palmivora is one of the most destructive phytopathogens affecting many economically important tropical crops such as coconut. P. palmivora causes diseases in over 200 host plants, and notably, the bud rot disease in coconut and oil palm, which is often lethal because it is usually detected at advanced stages of infection. Limited availability of large-scale omics datasets for P. palmivora is an important barrier for progress toward phytopathogen diagnostics. We report here the mycelial proteome of P. palmivora using high-resolution mass spectrometry analysis. We identified 8073 proteins in the mycelium. Gene Ontology-based functional classification of detected proteins revealed 4884, 4981, and 3044 proteins, respectively, with roles in biological processes, molecular functions, and cellular components. Proteins such as P-loop, NTPase, and WD40 domains with key roles in signal transduction pathways were identified. KEGG pathway analysis annotated 2467 proteins to various signaling pathways, such as phosphatidylinositol, Ca2+, and mitogen-activated protein kinase, and autophagy and cell cycle. These molecular substrates might possess vital roles in filamentous growth, sporangia formation, degradation of damaged cellular content, and recycling of nutrients in P. palmivora. This large-scale proteomics data and analyses pave the way for new insights on biology, genome annotation, and vegetative growth of the important plant pathogen P. palmivora. They also can help accelerate research on future phytopathogen diagnostics and preventive interventions.

Identifying Novel Genes and Proteins Involved in Salt Stress Perception and Signaling of Rice Seedlings.

Rice is one of the most important crops worldwide. Crop production is constrained markedly, however, by abiotic stresses such as salinity. To elucidate early stress response signaling networks involved in rice, we report in this study an original quantitative proteomic analysis of the rice seedlings subjected to short-term salt stress. We detected 570 differentially regulated proteins (DRPs) in the root sample. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that DRPs of the root were mainly involved in membrane trafficking, kinase activity, and ion toxicity responses. Interactome analysis revealed the central role of root proteins involved in membrane trafficking in the early response to salinity, such as cell surface receptor-like kinases (RLKs), phosphatidylinositols (PIs), calcium-dependent protein kinases 1 and 5, calcineurin B-like protein-interacting proteins, protein phosphatase 2C (PP2C) inhibitors, and abscisic acid receptors (PYL5/10), indicating activation of S-type anion channel. Furthermore, the proteogenomic analysis revealed 128 unique genome search-specific peptides with high-quality mass spectromety (MS/MS) spectra. We identified 38 novel protein-coding genes, refined the annotation of 17 existing gene models, and suggested several novel stress-responsive proteins, such as RLK5, peroxidase 27, and growth-regulating factor 2. Novel peptides had an ortholog match in the curated protein sequence set of other plant species. In conclusion, this study identifies novel stress-responsive proteins and genes of rice, thus warrant future consideration as candidates for molecular breeding of stress-tolerant crop varieties.

Unraveling Toxoplasma gondii GT1 Strain Virulence and New Protein-Coding Genes with Proteogenomic Analyses.

Toxoplasma gondii is one of the most widespread parasites of great relevance to planetary health. It infects approximately one-third of the world population. T. gondii establishes itself in warm-blooded animals and causes adverse health outcomes, particularly in immunocompromised patients. T. gondii is also widely used as a model organism to study other related apicomplexan parasites, which requires a deeper understanding of its molecular biology. Type I strains (GT1 and RH) of T. gondii are considered the most virulent forms. The whole-genome sequencing of T. gondii annotated 8460 predicted gene models in the parasite. To this end, the proteogenomics technology allows harnessing of mass spectrometry (MS)-derived proteomic data to unravel new protein-coding genes, not to mention validation and correction of the existing gene models. In this study using the proteogenomic approach, we report the identification of 31 novel protein-coding genes while reannotating 88 existing gene models. Notably, the genome annotations were corrected for genes, such as SAG5C, GRA6, ROP4, ROP5, and ROP26. The associated proteins are known to play important roles in host-parasite interactions, particularly in relation to parasite virulence, suppression of host immune response, and distinctively pertinent for the survival of the parasite inside the host system. These new findings offer new insights, informing planetary health broadly and the knowledge base on T. gondii virulence specifically. The proteogenomics approach also provides a concrete example to study related apicomplexan organisms of relevance to planetary health, and so as to develop new diagnostics and therapeutics against toxoplasmosis and related diseases.

Phosphorylation-mediated signalling in flowering: prospects and retrospects of phosphoproteomics in crops.

Protein phosphorylation is a major post-translational modification, regulating protein function, stability, and subcellular localization. To date, annotated phosphorylation data are available mainly for model organisms and humans, despite the economic importance of crop species and their large kinomes. Our understanding of the phospho-regulation of flowering in relation to the biology and interaction between the pollen and pistil is still significantly lagging, limiting our knowledge on kinase signalling and its potential applications to crop production. To address this gap, we bring together relevant literature that were previously disconnected to present an overview of the roles of phosphoproteomic signalling pathways in modulating molecular and cellular regulation within specific tissues at different morphological stages of flowering. This review is intended to stimulate research, with the potential to increase crop productivity by providing a platform for novel molecular tools.

Proteomic responses to progressive dehydration stress in leaves of chickpea seedlings.

BACKGROUND: Chickpea is an important food legume crop with high protein levels that is widely grown in rainfed areas prone to drought stress. Using an integrated approach, we describe the relative changes in some physiological parameters and the proteome of a drought-tolerant (MCC537, T) and drought-sensitive (MCC806, S) chickpea genotype. RESULTS: Under progressive dehydration stress, the T genotype relied on a higher relative leaf water content after 3 and 5 d (69.7 and 49.3%) than the S genotype (59.7 and 40.3%) to maintain photosynthetic activities and improve endurance under stress. This may have been facilitated by greater proline accumulation in the T genotype than the S genotype (14.3 and 11.1 µmol g- 1 FW at 5 d, respectively). Moreover, the T genotype had less electrolyte leakage and lower malondialdehyde contents than the S genotype under dehydration stress, indicating greater membrane stability and thus greater dehydration tolerance. The proteomic analysis further confirmed that, in response to dehydration, the T genotype activated more proteins related to photosynthesis, stress response, protein synthesis and degradation, and gene transcription and signaling than the S genotype. Of the time-point dependent proteins, the largest difference in protein abundance occurred at 5 d, with 29 spots increasing in the T genotype and 30 spots decreasing in the S genotype. Some of the identified proteins-including RuBisCo, ATP synthase, carbonic anhydrase, psbP domain-containing protein, L-ascorbate peroxidase, 6-phosphogluconate dehydrogenase, elongation factor Tu, zinc metalloprotease FTSH 2, ribonucleoproteins and auxin-binding protein-may play a functional role in drought tolerance in chickpea. CONCLUSIONS: This study highlights the significance of genotype- and time-specific proteins associated with dehydration stress and identifies potential resources for molecular drought tolerance improvement in chickpea.

Comparative proteomics and gene expression analyses revealed responsive proteins and mechanisms for salt tolerance in chickpea genotypes.

BACKGROUND: Salinity is a major abiotic stress that limits the growth, productivity, and geographical distribution of plants. A comparative proteomics and gene expression analysis was performed to better understand salinity tolerance mechanisms in chickpea. RESULTS: Ten days of NaCl treatments resulted in the differential expression of 364 reproducible spots in seedlings of two contrasting chickpea genotypes, Flip 97-43c (salt tolerant, T1) and Flip 97-196c (salt susceptible, S1). Notably, after 3 days of salinity, 80% of the identified proteins in T1 were upregulated, while only 41% in S2 had higher expression than the controls. The proteins were classified into eight functional categories, and three groups of co-expression profile. The second co-expressed group of proteins had higher and/or stable expression in T1, relative to S2, suggesting coordinated regulation and the importance of some processes involved in salinity acclimation. This group was mainly enriched in proteins associated with photosynthesis (39%; viz. chlorophyll a-b binding protein, oxygen-evolving enhancer protein, ATP synthase, RuBisCO subunits, carbonic anhydrase, and fructose-bisphosphate aldolase), stress responsiveness (21%; viz. heat shock 70 kDa protein, 20 kDa chaperonin, LEA-2 and ascorbate peroxidase), and protein synthesis and degradation (14%; viz. zinc metalloprotease FTSH 2 and elongation factor Tu). Thus, the levels and/or early and late responses in the activation of targeted proteins explained the variation in salinity tolerance between genotypes. Furthermore, T1 recorded more correlations between the targeted transcripts and their corresponding protein expression profiles than S2. CONCLUSIONS: This study provides insight into the proteomic basis of a salt-tolerance mechanism in chickpea, and offers unexpected and poorly understood molecular resources as reliable starting points for further dissection.