Ribosomal proteins are involved in the regulation of plant growth and development. However, the regulatory processes of most ribosomal proteins remain unclear. In this study, Arabidopsis plants with the mutation in ribosomal phosphoprotein P1A (RPP1A) produce larger and heavier seeds than wild-type plants. A comparative quantitative label-free proteomic analysis revealed that a total of 215 proteins were differentially accumulated between the young siliques of the wild type and rpp1a mutant. Knockout of RPP1A significantly reduced the abundance of proteins involved in carboxylic acid metabolism and lipid biosynthesis. Consistent with this, a metabolic analysis showed that the organic acids in the tricarboxylic acid cycle and the carbohydrates in the pentose phosphate pathway were severely reduced in the mature rpp1a mutant seeds. In contrast, the abundance of proteins related to seed maturation, especially seed storage proteins, was markedly increased during seed development. Indeed, seed storage proteins were accumulated in the mature rpp1a mutant seeds, and the seed nitrogen and sulfur contents were also increased. These results indicate that more carbon intermediates probably enter the nitrogen flow for the enhanced synthesis of seed storage proteins, which might subsequently contribute to the enlarged seed size in the rpp1a mutant. SIGNIFICANCE: Ribosomes are responsible for protein synthesis and are generally perceived as the housekeeping components in the cells. In this study, the knockout of RPP1A leads to an increased seed size through repressing carbon metabolism and lipid biosynthesis, and increasing the synthesis of seed storage proteins. Meanwhile, the abundance of seed storage proteins and the nitrogen and sulfur concentrations were increased in the mature rpp1a mutant seeds. The results provide a novel insight into the genetic regulatory networks for the control of seed size and seed storage protein accumulation, and this knowledge may facilitate the improvement of crop seed size.

Lysine acetylation (Kac) on histone promotes relaxation of the chromatin conformation and favors gene transcription to regulate oncogenesis, whereas the total acetylation profiling of oral squamous cell carcinoma (OSCC) is unknown. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilised to investigate lysine acetylation features of tumor tissues and adjacent normal tissues from 9 patients with OCSS. 282 upregulated Kac sites in 234 proteins and 235 downregulated Kac sites in 162 proteins between OSCC tissues and paired adjacent normal tissues were identified. Different acetylation proteins (DAPs) were analyzed through KEGG-based and MCODE. These DAPs are enriched in the ribosome biogenesis pathway. Survival Analysis of hub genes with TCGA database was performed. In addition, IPA software was used to explore the connection between 9 core DAPs (RPS3, RPL24, RPL19, EIF4A2, RPL12, MYBPC1, RPS6, ARCN1, and TMEM9) and the different expression of KATs and KDACs identified in our proteomic. The study is the first comparative study of Kac modification on oral squamous cell carcinoma. We propose to put forward the hypothesis that the dysfunction of ribosome biogenesis caused by the change of Lysine acetylation, especially downregulated acetylation on RPS6 and RPS3 may associated with the pathogenesis of OSCC. SIGNIFICANCE: The study is the first comparative study of Kac modification on oral squamous cell carcinoma through LC-MS/MS-based modified proteomic. These DAPs are high enriched in the ribosome biogenesis pathway. Used MCODE and survival analysis, 9 core DAPs (RPS3, RPL24, RPL19, EIF4A2, RPL12, MYBPC1, RPS6, ARCN1, and TMEM9) were screened. IPA software was used to explore the connection between 9 core DAPs and the different expression of KATs and KDACs identified in our proteomic. In addition, we propose to put forward the hypothesis that the dysfunction of ribosome biogenesis caused by the change of Lysine acetylation, especially downregulated acetylation on RPS6 and RPS3 may associated with the pathogenesis of OSCC.

Surgery, radiation therapy (RT), and chemotherapy are commonly used treatment modalities for CRC management. The locally advanced CRC is managed with preoperative RT or in combination of chemoradiotherapy whereas palliative RT is recommended for metastatic CRC patients to enhance overall survival and reduce distressing symptoms. There are many biomarkers established based on tumour staging, grading and molecular characteristics of patients (e.g., mutation, DNA methylation, and gene expression profiling). Interestingly, none of these markers are adequately validated for RT scheme. In order to establish the radioresponsive biomarker in CRC, we established a mouse xenograft tumour model and applied radiation to the tumours. We identified 9 metabolic proteins, namely PGK1, PGAM1, ENO1, PKM, TKT, GLUD1, LDHA, GAPDH, and MDH2, which are differentially expressed in tumours with different radioresponsiveness. Furthermore, we validated their expression in tumours from the unirradiated, poorly responded and highly responded tumour groups. In addition, we analysed their expressions in clinical samples from the public database. Extensive literature studies shown that these metabolic proteins are associated with key biochemical pathways including, glycolysis, ammonia detoxification, carcinogenesis, and drug responses. Further studies are needed to translate our findings into clinical use. SIGNIFICANCE: With the increasing incidence of colorectal cancer (CRC) globally, it is crucial to establish strategic treatment protocol by personalizing cancer treatment. Despite the well-established treatment protocols for CRC in the past decades, the mortality remains high. There is a trend of applying personalized treatment to improve patient survival. It has been reported that biomarkers may be used to predict treatment outcomes or to adjust individual treatment protocols. This project aims to identify specific metabolic proteins as biomarkers for CRC radioresponsiveness. Using bioinformatical analysis, we have identified 9 metabolic proteins which could be used as potential biomarkers for radiation therapy in CRC tumours.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease of unknown etiology in which the posttranslational modifications (PTMs) of proteins play an important role. PTMs, such as those involved in the formation of neutrophil extracellular traps (NETs), have been well studied. The excessive formation and release of NETs can mediate inflammation and joint destruction in RA. It has been gradually recognized that lysine malonylation (Kmal) can regulate some biological processes in some prokaryotes and eukaryotes. However, less is known about the role of Kmal in RA. We therefore performed proteome and malonylome analyses to explore the proteomic characteristics of the peripheral blood mononuclear cells from 36 RA patients and 82 healthy subjects. In total, 938 differentially expressed proteins (DEPs) and 42 differentially malonylated proteins (DMPs) with 55 Kmal sites were detected through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analysis. Functional analysis showed that two DEPs with four malonylated sites and one DMP with a malonylated site were identified in the neutrophil extracellular trap formation (NETosis) pathway. Altogether, this study not only describes the characteristics of the malonylome in RA for the first time, but it also reveals that malonylation may be involved in the NETosis pathway. SIGNIFICANCE: This is the first report that reveals the proteomic features of Kmal in RA through a LC-MS/MS-based method. In this study, we found that several key DMPs were associated with the NETosis pathway, which contributes to the development of RA. The present results provide an informative dataset for the future exploration of Kmal in RA.

Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. SIGNIFICANCE: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

Sepsis-induced myocardial depression is common among patients in the intensive care unit; however, the exact mechanisms underlying this condition remain unclear. We investigated differences in the expression of specific proteins and determined the potential functions of the proteins in a rat model of lipopolysaccharide-induced septic shock. Left ventricular tissue was excised from 16 rats (sepsis group, 8; control group, 8) and analysed. Quantitative analysis of the global proteome was performed using 4D label-free technique. Bioinformatic analyses were conducted based on differentially expressed (DE) proteins. Parallel reaction monitoring (PRM) validation for selected proteins and western blotting for selected global protein modifications in heart tissues were also performed. As a result, out of 3653 proteins identified, 108 were expressed differentially between the two groups. The bioinformatic analyses revealed that DE proteins play important roles in metabolism- and immune-related pathways. PRM results supported the plausibility and reliability of the proteomics data. Modification of heart tissue acetyllysine, succinyllysine, 2-hydroxyisobutyryllysine, and lactyllysine revealed clear differences between the two groups, indicating the effects of protein modification. Our study suggested that expression patterns of global proteins in heart tissue were different between the two groups. These results provide new valuable information on the possible mechanisms underlying sepsis-induced myocardial depression. SIGNIFICANCE: The expression patterns of global proteins in the heart tissues of patients with sepsis and control groups remain unknown. In this study, we used the 4D label-free proteomics technique to compare differentially expressed (DE) proteins between the sepsis and control groups. We identified 3653 proteins, 108 of which were expressed differentially between the sepsis and control groups. Further bioinformatic analyses revealed that DE proteins play critical roles in metabolism- and immune-related processes and pathways. Interestingly, modification of heart tissue acetyllysine, succinyllysine, 2-hydroxyisobutyryllysine, and lactyllysine revealed clear differences between the sepsis and control groups. The findings of this study improve our understanding of the basic molecular mechanisms underlying sepsis-induced myocardial depression.

The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14 (RPN11), USP14 and UCH37 (UCHL5). However, the functional roles and specificities of these proteasomal DUBs remain elusive. To reveal the specificities of proteasome associated DUBs, we used SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. We observed distinct effects on the global ubiquitinome upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggested less functional redundancy than previously anticipated. We also investigated whether the small molecule inhibitor b-AP15 has the potential to specifically target USP14 and UCH37 by comparing treatment of wild-type versus USP14/UCH37 double-knockout cells with this drug. Strikingly, broad and severe off-target effects were observed, questioning the alleged specificity of this inhibitor. In conclusion, this work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds. SIGNIFICANCE: Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive. MATERIALS & METHODS: We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37. RESULTS: We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor. CONCLUSIONS: This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.

Phosphorylation is a posttranslational modification of proteins that regulates many cellular processes, such as communication between cells, cell proliferation, cell movements, and gene expression. Therefore, many studies have been conducted to determine the significance and function of phosphorylation. These studies involve the identification of phosphorylation site(s), kinases and phosphatases, and regulatory mechanisms. Recently, phosphorylation sites were identified using mass spectrometry and detected by immunoblotting with phosphorylation site-specific antibodies. However, the in vivo phosphorylation profile of the target protein is not easy to grasp, and the quantification of site-specific phosphorylation is challenging if the protein is phosphorylated at multiple sites. Phos-tag is a phospho-affinity SDS-PAGE approach in which phosphorylated proteins are separated depending on the number and sites of phosphorylation during electrophoresis, which overcomes the aforementioned problems. We applied this technique to perform an in vivo analysis of the phosphorylation of many proteins. In this article, we show our results for the phosphorylation of tau protein, p35 Cdk5 activator and GSK3β to reveal the utility and power of this technique in protein phosphorylation analyses in vivo. SIGNIFICANT: We show the in vivo phosphorylation of tau and two tau kinases analysed by using Phos-tag SDS-PAGE. Tau represents about 12 different phosphoisotypes when expressed in cultured cells. Tau is differently phosphorylated in patients with different tauopathy. Phosphorylation of p35 Cdk5 activator, which suppress the abnormal activation of Cdk5 by cleavage with calpain, is regulated developmentally. The Ser9 phosphorylation is not a proper marker of the GSK3β activity in vivo.

Leptospirosis is a global zoonotic disease affecting humans and livestock species. Bacterin vaccines lack cross protection between serogroups, and include multiple serovars propagated at 29 °C. Recent work demonstrated substantial variation in the transcriptome of identical species and serovars of Leptospira. Here, substantial differences in protein abundance profiles were identified in Leptospira borgpetersenii serovar Hardjo; strain HB203, which was isolated in the 1980s, compared to newer strains TC129 and TC273 isolated in 2016, and whether they were propagated at the routine temperature of 29 °C, compared to 37 °C which more closely emulates host infection. While 388 and 385 significantly differentially expressed (DE) proteins (FDR of 0.01) were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 29 °C respectively, only 66 and 4 DE proteins were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 37 °C respectively. Within each strain comparing temperatures, HB203 had 524 significantly DE proteins, TC129 had 347 DE proteins, and TC273 had 569 DE proteins. Data are available via ProteomeXchange with identifier PXD032831. Results highlight significant differential protein expression among identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth. SIGNIFICANCE: Leptospirosis is a zoonotic disease caused by spirochete bacteria of the genus Leptospira. While leptospirosis affects over one million people per year, symptoms range vastly in severity from completely asymptomatic, to flu-like, to multi-organ failure and death in severe cases. Incidental hosts become infected after encountering pathogens directly from contact with another host, including domestic or wildlife animals, or indirectly from contaminated environments. Though animal vaccines exist, they lack cross protection across serogroups, and instead rely on inclusion of multiple carefully selected serovars from laboratory strains prepared at ~29 °C. Recent interest in gene expression at the Leptospira strain level, along with a newly achieved culture temperature of 37 °C (which more closely resembles host body temperature), led us to investigate the proteomic profiles of an older, established challenge strain HB203 in comparison to TC129 and TC273, two strains isolated in 2016 from abattoir cattle in the central United States. Herein, we identify substantial proteomic differences not only between strains of the same species and serovar, but notably between growth temperatures, collectively suggesting that bacterin vaccine composition may benefit from investigating strain selection and the temperature employed for growth of the bacteria used in bacterin preparation.

Capsicum belonging to the family Solanaceae is one of the most widely consumed crops in the world as a vegetable, spice and a raw salad and is distinctly valuable for its spicy pungent flavour. Proteomic investigation of crop plants is an essential step towards deciphering the functional basis of traits in an organism and to deepen our understanding on the regulation of various developmental patterns, biotic, and abiotic stress response and tolerance mechanisms. The differential proteome expression profiling of tissues during different developmental stages and under different conditions may indicate the specific proteome dynamics involved in the developmental programs and under stress conditions. Although substantial progress in proteomics of other Solanaceae plants has been made in the past two decades, a comprehensive review on Capsicum proteomics is still lacking. This review provides updated information on the advancement of Capsicum proteomic study in cytoplasmic male sterility, during fruit development and ripening, and under different biotic and abiotic stresses. Although limited information is available on the post translational protein modifications in Capsicum, a brief outline is given at the end detailing various post translational modifications. This proteomic update on Capsicum will be useful for future studies aimed at Capsicum improvement programs.

Phosphorylation is an essential regulatory mechanism in cells that modifies diverse substrates, such as proteins, carbohydrates, lipids, and nucleotides. Protein phosphorylation regulates function, subcellular localization, and protein-protein interactions. Protein kinases and phosphatases catalyze this reversible mechanism, subsequently influencing signal transduction. The dysregulation of protein phosphorylation leads to many diseases, such as cancer, neurodegenerative diseases, and metabolic diseases. Therefore, analyzing the phosphorylation status and identifying protein phosphorylation sites are critical for elucidating the biological functions of specific phosphorylation events. Unraveling the critical phosphorylation events associated with diseases and specific signaling pathways is promising for drug discovery. To date, highly accurate and sensitive approaches have been developed to detect the phosphorylation status of proteins. In this review, we discuss the application of Phos-tag to elucidate the biological functions of Hippo pathway components, with emphasis on the identification and quantitation of protein phosphorylation under physiological and pathological conditions. SIGNIFICANCE: We here provide a comprehensive overview of Phos-tag technique-based strategies to identify phosphorylated proteins at the cellular level in the Hippo-YAP pathway that comprises a major driving force for cellular homeostasis. We clarify the links of applying Phos-tag in elucidating the biological functions of the Hippo pathway components with particular attention to the identification and quantitation of protein phosphorylation under physiological and pathological conditions. We believe that our paper will make a significant contribution to the literature because these detailed phosphorylation modifications and functional diversity of the Hippo pathway components in physiological and pathological processes are only beginning to come to the fore, highlighting the potential for discovering new therapeutic targets. Moreover, this line of research can provide further insight into the inextricable link between phos-tag applications as a molecular tool and cellular signaling modality, offering new directions for an integrated research program toward understanding cellular regulation at the molecular level. Given the broad research and practical applications, we believe that this paper will be of interest to the readership of your journal.

The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Proteins in the uterine luminal fluid are essential for embryo development and regulation of embryo-maternal interaction in porcine. However, little is known about the profile of proteins in uterine luminal fluid of porcine during the pre-implantation period. The present study, applied iTRAQ proteomics technology to identify and analyze uterine luminal fluid proteins on day 9 of estrus cycle and days 9, 12, and 15 of pregnancy. A total of 964 proteins were identified in the present study. Principal component analysis and hierarchical clustering revealed the dynamic developmental characteristics of embryo implantation, which indicated significant differences on day 12 or 15 of pregnancy. In addition, further analysis conducted in the present study identified 279 differentially abundance proteins among the three groups, five clusters were generated using SOTA clustering to examine changes in of the differentially abundant proteins. Results of the current study also found that the proteins in the cluster are involved in some important processes such as regulation of low-density lipoproteins and regulation of TGF-β secretion. Notably, it was found that regulation of TGF-β is essential for porcine embryonic morphological transformation. Furthermore, proteins that play vital roles in implantation, such as CTSC, CTSB, and ACP5 were identified through protein-protein interaction network. Therefore, these findings of the present study provide a basis for understanding embryo development mechanisms and implantation in pigs. SIGNIFICANCE: Proteins are directly acting molecules for the functioning of organisms. It is important to study the regulation mechanism of embryo implantation from the perspective of protein function. In the current study, iTRAQ proteomics technology was employed to identify and explore uterine luminal fluid proteins on day 9 of estrus cycle and days 9, 12, and 15 of pregnancy. The findings provide novel insights into the process of porcine early embryo implantation. Furthermore, it is also helpful to clarify the mechanism of embryonic development and implantation.

Cowpea (Vigna unguiculata L. Walp) is a legume of great economic importance, however it is highly affected by nematodes. The present work aimed to identify proteins and genes involved in nematode resistance by proteomic and transcriptomic analysis. Plants of a genotype resistant (CE31) to root-knot nematode (Meloidogyne spp.) were collected 12 days after inoculation with Meloidogyne incognita and the total proteins and RNA were extracted from the root samples. Shotgun proteomic analysis was performed using an Orbitrap Elite mass spectrometer and the construction and sequencing of cDNA libraries were carried out in a Hi-Seq 2000 sequencing system. The proteomic and transcriptomic analyses revealed key processes involved in cowpea defense and some interesting candidates were further analyzed by RT-qPCR. Proteins and genes involved in essential biological processes were differentially accumulated such as, regulation of transcription, cell wall stiffening and microtubule-based process. However, the main defense strategies of Vigna unguiculata seem to be focused on the interaction of NBS-LRR and WRKY genes for the activation of R genes, production of protease inhibitors and maintenance of actin cytoskeleton. These are key processes that can culminate in the suppression of giant cell formation and consequently in the development of Meloidogyne incognita. SIGNIFICANCE: In this study, we identified proteins and transcripts regulated in cowpea resistant to the nematode Meloidogyne spp. upon inoculation. The results revealed key candidate genes involved in the activation of R genes, the production of protease inhibitors and maintenance of the actin cytoskeleton. These processes might be essential for cowpea resistance, as they can impede nematode nutrition, giant cell formation and consequently the development of Meloidogyne incognita.

The Asian citrus psyllid, Diaphorina citri, is the vector of Candidatus Liberibacter asiaticus (CLas), the presumed causative agent of citrus greening disease. For successful transmission, CLas must cross the gut barrier, requiring interaction with proteins on the midgut epithelium. We compared the relative abundance of gut surface proteins for both adult and nymph D. citri, as nymphs are particularly susceptible to CLas infection. To enrich for gut surface proteins, brush border membrane vesicles were prepared from dissected guts, and proteins identified from triplicate samples run on a timsTOF mass spectrometer. A total of 1516 and 1219 proteins were identified from D. citri adults and nymphs respectively. Based on bioinformatics analysis software and manual curation, 112 adult and 87 nymph proteins were predicted to localize to the surface of the microvilli and were further categorized into integral membrane and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteins exploited by insect pathogens such as aminopeptidase, alkaline phosphatase, cadherin, ABC transporters, and carboxypeptidase were among the most abundant proteins on the gut surface. In addition to providing insights into hemipteran gut physiology, the D. citri gut surface proteome will inform novel approaches to interfere with CLas interaction with the psyllid gut to prevent the spread of citrus greening. BIOLOGICAL SIGNIFICANCE: The Asian citrus psyllid (ACP), D. citri is one of the most serious pests of citrus worldwide. ACP transmits the pathogenic bacterium that causes citrus greening or huanglongbing (HLB), which has resulted in severe economic losses in global citriculture. The putative causative agent of this disease, the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), is vectored by the Asian citrus psyllid, D. citri, in a persistent and circulative manner. CLas must interact with gut surface proteins in order to enter midgut epithelial cells. However, the specific proteins exploited by CLas have yet to be identified. The characterization of the most abundant proteins on the surface of the D. citri gut provides insight into candidate receptors for CLas and other pathogens of D. citri. We hypothesize that pathogens of D. citri exploit the most abundant proteins on the surface of the gut for entry into the host insect. Importantly, the abundant gut surface proteins will provide the basis for novel approaches to disrupt CLas-D. citri interactions, with the goal of preventing further economic loss to the citrus industry.

Septic arthritis (SA) is a life-threatening condition in horses, and identifying eradication of infection in equine SA is challenging. This study explored the discovery of putative biomarkers for the eradication of joint infection in horses. We performed proteomics analysis of synovial fluid (SF) and plasma from horses with experimental SA, non-septic lipopolysaccharide-induced arthritis, and controls. The point of eradication of infection in horses with SA was determined previously. We compared spectral intensities between groups as well as before and after the eradication of infection. Twenty-six differentially abundant proteins were identified, which were upregulated in SF of horses with SA compared to the other groups, as well as compared to the same horses post-eradication of infection. In plasma, we did not identify differentially abundant proteins. Differentially abundant proteins in SF were of cellular origin and their biological functions included ubiquitination, signal transduction, apoptosis etc. The difference in their relative abundance between experimental groups was ≥10-fold compared to the abundance expected based on the difference in cell count alone (2-fold). Since most of cells in joints with bacterial infection are neutrophils, we suggest that the variable abundance of neutrophil- and cell-associated proteins represent potential biomarkers of eradication of infection in equine SA. SIGNIFICANCE: Septic arthritis is an important condition in horses, which can be life-threatening. At present, identifying eradication of infection in cases of equine septic arthritis is challenging. In this study, we performed a global proteomics analysis of synovial fluid and plasma in horses with experimental septic arthritis and identified 26 differentially abundant proteins compared to non-septic arthritis and post eradication of infection. The results of this study provide the basis for further characterization of the differentially abundant proteins and identification of clinically relevant biomarkers of septic arthritis in horses.

We have developed a family of QconCAT standards for the absolute quantification of pharmacological target proteins in a variety of human tissues. The QconCATs consist of concatenated proteotypic peptides, are designed in silico, and expressed in E. coli in media enriched with [13C6] arginine and [13C6] lysine to generate stable isotope-labeled multiplexed absolute quantification standards. The so-called MetCAT (used to quantify cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes), the liver TransCAT (used to quantify plasma-membrane drug transporters) and the brain TransCAT (used to quantify transporters expressed in the blood-brain barrier) were previously reported. We now report new QconCATs for the quantification of non-UGT non-CYP drug metabolizing enzymes (NuncCAT) and receptor tyrosine kinases (KinCAT). We have also redesigned the liver TransCAT, replacing problematic peptides and the N-terminal tag, for better characterization and expression. All these QconCATs showed high purity, high labelling efficiency with stable 13C isotope (>95%), and high sequence coverage (>88%). They represent a close-knit family of standards for quantifying pharmacokinetic targets, together with a more distant cousin, the KinCAT, used to quantify pharmacodynamic targets. SIGNIFICANCE: Multiplexed determination of absolute protein abundances using quantitative conCATemers (QconCATs) has already been successfully demonstrated in different human tissues. We have previously reported two QconCATs; MetCAT and TransCAT, for the quantification of key enzymes (cytochrome P450 enzymes (CYP) and glucuronosyltransferases (UGT)) and drug transporters. To build on these reports, application of the QconCAT methodology for the determination of non-UGT non-CYP enzymes and receptor tyrosine kinases (RTKs) in human tissue is reported here. This report focuses on development and characterization of two QconCAT constructs for the quantification of 24 enzymes and 21 RTKs. We demonstrate that the developed QconCATs have high purity, high incorporation efficiency and low peptide miscleavage upon proteolysis. Application of these QconCATs for reliable quantification of target proteins was achieved in human liver.

Male sterile mutants can be used in breeding or commercial cultivation in tomato, but there are few research reports on their proteomics. In this study, we analyzed the metabolic pathways and biological functions of differentially abundant proteins (DAPs) involved in two stages of stamen development of the tomato flowers by using a high through-put iTRAQ labeled proteomic approach. There was a total of 1476 DAPs which should associated with the occurrence of pollen abortion in tomato. Moreover, there were more DAPs in the four membrane systems. It shows that membrane systems are very important for tomato pollen development. According to KEGG analysis, these signaling pathways including starch and sucrose metabolism (map00500), tropane, piperidine and pyridine alkaloids biosynthesis (map00960), amino sugar and nucleotide sugar metabolism (map00520) have important effects on pollen development. These results were verified by using mass spectrometry PRM. Finally, two candidate genes (Solyc11g065770 and Solyc11g065530) were found that may be related to pollen development and cause pollen abortion by comparison of protein-protein interaction networks and on the basis of previous studies on ms-7 gene. This data and model will provide a new insight into tomato genetic male sterility 7 and contribute to the improvement of tomato hybrid breeding. Biological significance: Artificial emasculation is still the main method of tomato hybrid breeding at present. Adopting male sterility in tomato cross breeding could greatly improve the production efficiency and seed purity; reduce the cost. Although numerous researches have been conducted to select the genes related to male sterility, the molecular mechanism remains unclear in tomato. In this study, we used the high-through-put iTRAQ labeled proteomic approach, to perform a novel comparison of expression profiles in GMS tomato line and its wildtype line. Based on these results, we proposed the potential regulated protein network involved in pollen development mechanism of tomato GMS and two candidate genes. SIGNIFICANCE: Artificial emasculation is still the main method of tomato hybrid breeding at present. Adopting male sterility in tomato cross breeding could greatly improve the production efficiency and seed purity; reduce the cost. Although numerous researches have been conducted to select the genes related to male sterility, the molecular mechanism remains unclear in tomato. In this study, we used the high-through-put iTRAQ labeled proteomic approach, to perform a novel comparison of expression profiles in GMS tomato line and its wildtype line. Based on these results, we proposed the potential regulated protein network involved in pollen development mechanism of tomato GMS and two candidate genes.

Understanding common and distinct pathophysiological features between acute progressive ischemic stroke (APIS) and acute non-progressive ischemic stroke (ANPIS) is a prerequisite to making clear the mechanism to determine the prognosis of acute ischemic stroke (AIS). Here, we recruited three independent sets of subjects, all of which included the APIS, ANPIS, and control groups. They were used for serum proteomic and metabolomic analyses, and validation of the critical pathophysiological processes and potential biomarkers of APIS, respectively. Results showed that there were both common and distinct metabolome and proteome between APIS and ANPIS. APIS and ANPIS shared basic processes of AIS in inflammation and oxidative stress response. Coagulation and lipid metabolism disorder, activation of the complement system, and inflammation may enhance with each other in the symptom worsening of APIS. The contents of serum amyloid A1 (SAA1) and S100 calcium-binding protein A9 (S100-A9) in the validation set confirmed the key pathophysiological processes indicated by omics data; they also jointly conferred a moderate value to distinguish APIS from ANPIS. Collectively, disturbance in coagulation and lipid metabolism, complement activation, and inflammation may be synergistically involved in symptom deterioration in APIS. SAA1 and S100-A9 serve as a potential biomarker panel to distinguish APIS from ANPIS. THE SIGNIFICANCE: In this study, we integrated serum proteomics and metabolomics to explore the similarities and differences in pathophysiological processes between APIS and ANPIS. The global metabolic networks have been constructed, and the crucial common pathophysiological processes and the key distinct pathophysiological features between APIS and ANPIS were investigated based on the differentially expressed proteins and metabolites (DEPs/DEMs). Furthermore, pivotal serum proteins (SAA1 and S100A9) were detected in a dependent set to validate the key pathophysiological characteristics, as well as to assess the possibility of them being used as a biomarker panel. Taken together, the multi-omics integration strategy used in this clinical study shows potential to comprehensively interpret and compare the pathophysiological processes of AIS in various conditions, as well as to screen a reliable new biomarker panel.

The importance of obtaining comprehensive and accurate information from cellular proteomics experiments asks for a systematic investigation of sample preparation protocols. In particular when working with unicellular organisms with strong cell walls, such as found in the model organism and cell factory Saccharomyces cerevisiae. Here, we performed a systematic comparison of sample preparation protocols using a matrix of different conditions commonly applied in whole cell lysate, bottom-up proteomics experiments. The different protocols were evaluated for their overall fraction of identified spectra, proteome and amino acid sequence coverage, GO-term distribution and number of peptide modifications, by employing a combination of database and unrestricted modification search approaches. Ultimately, the best protocols enabled the identification of approximately 65-70% of all acquired fragmentation spectra, where additional de novo sequencing suggests that unidentified spectra were largely of too low spectral quality to provide confident spectrum matches. Generally, a range of peptide modifications could be linked to solvents, additives as well as filter materials. Most importantly, the use of moderate incubation temperatures and times circumvented excessive formation of modification artefacts. The collected protocols and large sets of mass spectrometric raw data provide a resource to evaluate and design new protocols and guide the analysis of (native) peptide modifications. SIGNIFICANCE: The single-celled eukaryote yeast is a widely used model organism for higher eukaryotes in which, for example, the regulation of glycolysis is studied in the context of health and disease. Moreover, yeast is a widely employed cell factory because it is one of the few eukaryotic organisms that can efficiently grow under both aerobic and anaerobic conditions. Large-scale proteomics studies have become increasingly important for single-celled model organisms, such as yeast, in order to provide fundamental understanding of their metabolic processes and proteome dynamics under changing environmental conditions. However, comprehensive and accurate cellular proteomics experiments require optimised sample preparation procedures, in particular when working with unicellular organisms with rigid cell walls, such as found in yeast. Protocols may substantially bias towards specific protein fractions, modify native protein modifications or introduce artificial modifications. That lowers the overall number of spectral identifications and challenges the study of native protein modifications. Therefore, we performed a systematic study of a large array of protocols on yeast grown under highly controlled conditions. The obtained outcomes, the collected protocols and the mass spectrometric raw data enable the selection of suitable sample preparation elements and furthermore support the evaluation of (native) peptide modifications in yeast, and beyond.

Bovine faecal composition is complex and a knowledge gap exists in the understanding of the bovine faecal proteome. In the present study, in-gel sample preparation (IGSP) of faecal samples prior to proteomics showed an increase in the number of proteins identified in faecal samples compared to those processed by filter-aided sample preparation (FASP). The optimised sample preparation method removed high molecular weight glycoproteins as part of the clean-up process of the faecal samples, and in combination with in-gel digestion before liquid chromatography with tandem mass spectrometry (LC-MS/MS). The use of IGSP led to enhanced protein identification with increases in the number of peptides identified and in the percent coverage of proteins in the bovine faecal samples. SIGNIFICANCE: Characterization of faecal proteins has the potential to increase our understanding of host responses to changes such as diet, disease and drug-treatment. In-gel sample preparation prior to proteomics can be used to remove high molecular weight glycoproteins and reduce protein/peptide loss in FASP. This method of sample preparation will have application not only in the investigation of bovine faecal extracts but also in studies where large molecules such as glycoproteins or oligosaccharides could have detrimental influences on sample preparation involving ultrafiltration.

The cAMP-protein kinase A (PKA) pathway in platelets is important for both platelet activation and inactivation. We hypothesize that proteins/processes downstream of the cAMP-PKA pathway that are regulated after platelet activation ánd subsequent inactivation can serve as a "switch" in platelet activation and inhibition. We used a STRING-based protein-protein interaction network from proteins of interest distilled from publicly available quantitative platelet proteome datasets. The protein network was integrated with biological pathway information by functional enrichment analysis, phosphorylation by PKA, and drug-target information. Functional enrichment analysis revealed biological processes related to vesicle secretion and cytoskeletal reorganization to be overrepresented among these 30 proteins coinciding with topological clusters in the network. Our method identified proteins/processes with functions related to vesicle transport, cyclin-dependent protein kinases, tight junctions, and small GTPases as potential switches in platelet activation and inhibition. Next to established enzymes in cAMP-PKA signaling, such as PDE3A, proteins with an unknown/less well-known role in platelet biology, such as Stonin-2 and ABLIM-3, emerged from our analysis as interesting candidates for reversal of platelet activation. Our method can be used to repurpose existing datasets and provide a coherent overview of mechanisms involved to predict novel connections, by visually integrating multiple datasets. SIGNIFICANCE: This article presents a novel approach of visually incorporating multiple existing tools and proteomics datasets and in doing so provides novel insight into the complex molecular mechanisms involved in platelet activation. Using our approach, we also highlight several interesting candidates for future research into pathologies with high platelet reactivity.