Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SIGNIFICANCE: Mastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

Erratum in J Proteomics. 2022 Jan 6;250:104390.

Nitrogen (N) fertilizer is essential to ensure grain yield and quality in bread wheat. Improving N use efficiency is therefore crucial for wheat grain protein quality. In the present work, we analysed the effects on the winter wheat grain proteome of biostimulants containing Glutacetine® or two derived formulations (VNT1 and 4) when mixed with urea-ammonium-nitrate fertilizer. A large-scale quantitative proteomics analysis of two wheat flour fractions produced a dataset of 4369 identified proteins. Quantitative analysis revealed 9, 39 and 96 proteins with a significant change in abundance after Glutacetine®, VNT1 and VNT4 treatments, respectively, with a common set of 11 proteins that were affected by two different biostimulants. The major effects impacted proteins involved in (i) protein synthesis regulation (mainly ribosomal and binding proteins), (ii) defence and responses to stresses (including chitin-binding protein, heat shock 70 kDa protein 1 and glutathione S-transferase proteins), (iii) storage functions related to gluten protein alpha-gliadins and starch synthase and (iv) seed development with proteins implicated in protease activity, energy machinery, and the C and N metabolism pathways. Altogether, our study showed that Glutacetine®, VNT1 and VNT4 biostimulants positively affected protein composition related to grain quality. Data are available via ProteomeXchange with identifier PXD021513. SIGNIFICANCE: We performed a large-scale quantitative proteomics study of the total protein extracts from flour samples to determine the effect of Glutacetine®-based biostimulants treatment on the protein composition of bread wheat grain. To our knowledge, only a few studies in the literature have applied proteomic approaches to study bread wheat grains and in particular to investigate the effect of biostimulants on the grain proteome of this cereal crop. In addition, most approaches used fractional extraction of proteins to target reserve proteins followed electrophoresis which leads to low identification rate of proteins. We identified and quantified a large protein dataset of 4369 proteins and determined ontological class of proteins affected by biostimulants treatments. Our proteomics investigation revealed the important role of these new biostimulants in achieving significant changes in protein synthesis regulation, storage functions, protease activity, energy machinery, C and N metabolism pathways and responses to biotic and abiotic stresses in grain.

Glycosylation affects clinical efficacy and safety; therefore, is a critical quality attribute of therapeutic monoclonal antibodies. Glycans are often labile and complex in patterns, giving rise to macro- and micro-heterogeneity. Recombinant production, diverse geographical locations, associated transportation and storage conditions further compound the problem. Two-way studies comparing glycoprofile of the originator and its given biosimilar are aplenty. However, the extent of analytical variation and similarity in glycoprofile across all approved versions of a drug is hardly explored. Using UHPLC and mass spectrometry, we compared the glycoprofiles of eight rituximab drug samples licensed for sale in India. While the types of glycans were found identical, the abundance of some glycans varied significantly within the tested population. The quality range of glycosylation parameters of the tested sample population differed significantly from the previously established values for US/EU licensed rituximab. As the mean abundance of the 90% of identified glycans falls within ±3SD, the extent of mutual variations amongst tested lots is less significant compared to the extreme deviation from previously established QR limits. Thus, we propose this approach as an orthogonal method to capture glycan variations in licensed versions of mAbs for quality surveillance and in cases where originator samples' are limiting. SIGNIFICANCE: As fluctuation in glycosylation may be of clinical significance, we identify that a one-to-one comparison with originator alone is insufficient in sensing the extent of variations in glycosylation parameters in licensed biosimilars of a given therapeutic mAb. Here we propose that future biosimilarity analysis may include an orthogonal approach of generating an additional combined QR range representing variations across the originator and its biosimilars. The glycosylation profiles of eight rituximab drug samples of different make obtained from the point of sale in India were found identical amongst the tested rituximab versions. However, the QR limits corresponding to important glycosylation parameters differed significantly across all tested samples from the previously established QR limits of US- and EU-licensed rituximab in statistical terms. Such an approach may be useful in defining the true range of glycan variations in licensed versions of therapeutic mAbs.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis's pathogenic hallmarks is its ability to persist in a dormant state in the host. Thus, this pathogen has developed mechanisms to withstand stressful conditions found in the human host. Particularly, the Ser/Thr-protein kinase PknG has gained relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomic analyses of protein extracts from M. tuberculosis H37Rv and a mutant lacking pknG. We found that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance compared with the wild-type. The main biological processes affected by pknG deletion were cell envelope components biosynthesis and response to hypoxia. Thirteen DosR-regulated proteins were underrepresented in the pknG deletion mutant, including Hrp-1, which was 12.5-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, our results allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival. SIGNIFICANCE: PknG is a Ser/Thr kinase from Mycobacterium tuberculosis with key roles in bacterial metabolism and bacterial survival within the host. However, at present the molecular mechanisms underlying these functions remain largely unknown. In this work, we evaluate the effect of pknG deletion on M. tuberculosis proteome using different approaches. Our results clearly show that the global proteome was remodeled in the absence of PknG and shed light on new molecular mechanism underlying PknG role. Altogether, this work contributes to a better understanding of the molecular bases of the adaptation of M. tuberculosis, one of the most deadly human pathogens, to its host.

Arthrospira platensis (Spirulina) is a microalga with a high content of crude protein. It has a recalcitrant cell wall that limits the accessibility of the animal endogenous enzymes to its intracellular nutrients. Enzymatic supplementation aiming to degrade cell walls could benefit microalgae digestibility. The objective of this study was to evaluate the impact of dietary Spirulina and lysozyme supplementation over the muscle proteome of piglets during the post-weaning stage. Thirty piglets were randomly distributed among three diets: control (no microalga), SP (10% Spirulina) and SP + L (10% Spirulina +0.01% lysozyme). After 4 weeks, they were sacrificed and samples of the longissimus lumborum muscle were taken. The muscle proteome was analysed using a Tandem Mass Tag (TMT)-based quantitative approach. A total of 832 proteins were identified. Three comparisons were computed: SP vs Ctrl, SP + L vs Ctrl and SP + L vs SP. They had ten, four and twelve differentially abundant proteins. Glycogen metabolism and nutrient reserves utilization are increased in the SP piglets. Structural muscle protein synthesis increased, causing higher energy requirements in SP + L piglets. Our results demonstrate the usefulness of proteomics to disclose the effect of dietary microalgae, whilst unveiling putative mechanisms derived from lysozyme supplementation. Data available via ProteomeXchange with identifier PXD024083. SIGNIFICANCE: Spirulina, a microalga, is an alternative to conventional crops which could enhance the environmental sustainability of animal production. Due to its recalcitrant cell wall, its use requires additional measures to prevent anti-nutritional effects on the feeding of piglets in the post-weaning period, during which they endure post-weaning stress. One of such measures could be CAZyme supplementation to help degrade the cell wall during digestion. Muscle proteomics provides insightful data on the effect of dietary microalgae and enzyme activity on piglet metabolism.

Protein ubiquitination is a dynamic post-translational modification involved in various biological processes in eukaryotes. To understand the function of ubiquitinated proteins in maize kernels, we used the specific K-GG antibody coupled with high-resolution LC-MS/MS to identify the ubiquitinated proteins in maize immature kernels. A total of 1999 lysine ubiquitination sites in 881 proteins were identified in maize kernels. Eight conserved ubiquitination motifs included KubD, GKub, EKub, KubXXXE, AKub, NXKub, KubXXXXXN, and KKub were found in ubiquitinated peptides. The ubiquitinated lysine neighborhoods are more frequently presented in ordered structures. Go and KEGG analysis showed the proteins involved in carbohydrate metabolism and protein processing were identified to be the targets of lysine ubiquitination. Other proteins, which related to RNA transport, spliceosome, endocytosis, ubiquitin-mediated proteolysis, proteasome, and MAPK signaling, were also found to be ubiquitinated. Protein-protein interaction network and KEGG analysis indicated that protein ubiquitination plays a major role in regulating many cellular processes and modulating diverse interactions in maize kernel development. The identification of the 881 ubiquitinated proteins in maize kernels provides a foundation for understanding the physiological roles of these ubiquitinated proteins. Our finding also provides a new insight view into the function of ubiquitinated proteins involved in maize kernel development. SIGNIFICANCE: We reported here the comprehensive proteomic analysis of the ubiquitin-modified proteome in maize kernel. We found that there are some new characteristics of them in ubiquitome of maize immature kernels. The results suggested that protein ubiquitination, as a post-translation modification, plays an essential role in regulating many cellular processes in maize kernel development. This study expands our knowledge on the regulatory roles and mechanisms of protein ubiquitination in maize. and other plants.

Chemoresistance is a major factor driving breast cancer (BC) relapse and the high rates of cancer-related deaths. Aberrant levels of glycans are closely correlated with chemoresistance. The essential functions of glycans in chemoresistance is not systematically studied. In this study, an integrated strategy with a combination of transcriptomics, proteomics, glycomics and glycoproteomics was applied to explore the dysregulation of glycogenes, glycan structures and glycoproteins in chemoresistance of breast cancer cells. In paclitaxel (PTX) resistant MCF7 cells, 19 differentially expressed N-glycan-related proteins were identified, of which MGAT4A was the most significantly down-regulated, consistent with decrease in MGAT4A expression at mRNA level in PTX treated BC cells. Glycomic analysis consistently revealed suppressed levels of multi-antennary branching structures using MALDI-TOF/TOF-MS and lectin microarray. Several target glycoproteins bearing suppressed levels of multi-antennary branching structures were identified, and ERK signaling pathway was strongly suppressed in PTX resistant MCF7 cells. Our findings demonstrated the aberrant levels of multi-antennary branching structures and their target glycoproteins on PTX resistance. Systematically integrative multi-omic analysis is expected to facilitate the discovery of the aberrant glycosyltransferases, N-glycosylation and glycoproteins in tumor progression and chemoresistance. SIGNIFICANCE: An integrated strategy with a combination of transcriptomics, proteomics, glycomics and glycoproteomics is crucial to understand the association between glycans and chemoresistance in BC. In this multi-omic analysis, we identified unique glycan-related protein, glycan and glycoprotein signatures defining PTX chemoresistance in BC. This study might provide valuable information to understand molecular mechanisms underlying chemoresistance in BC.

Histidine phosphorylation is critically important in a variety of cellular processes including signal transduction, cell cycle, proliferation, differentiation, and apoptosis. It is estimated to account for 6% of all phosphorylated amino acids. However, due to the acid lability of the PN bond, the study of pHis lags far behind that of pSer, pThr, and pTyr. Recently, the development and use of pHis-specific antibodies and methodologies have led to a resurgence in the study of histidine phosphorylation. Although a considerable number of pHis proteins and sites have been discovered, most of them have not been manually curated and integrated to any databases. There is a lack of a data repository for pHis, and such work is expected to help further systemic studies of pHis. Thus, we present a comprehensive resource database of histidine phosphorylation (HisPhosSite) by curating experimentally validated pHis proteins and sites and compiling putative pHis sites with ortholog search. HisPhosSite contains 776 verified pHis sites and 2702 verified pHis proteins in 38 eukaryotic and prokaryotic species and 15,378 putative pHis sites and 10,816 putative pHis proteins in 1366 species. HisPhosSite provides rich annotations of pHis sites and proteins and multiple search engines (including motif search and BLAST search) for users to locate pHis sites of interest. HisPhosSite is available at http://reprod.njmu.edu.cn/hisphossite. SIGNIFICANCE: Histidine phosphorylation is involved in a variety of cellular processes as well as cancers, and it has been proved to be more common than previously thought. The HisPhosSite database was developed to collect pHis data from published literatures with experimental evidences. Unification of the identified pHis proteins and sites will give researchers an informative resource for histidine phosphorylation. HisPhosSite has a user-friendly interface with multiple search engines for users to locate pHis sites of interest. In addition, the database provides rich structural and functional annotations. HisPhosSite will help future studies and elucidation of the functions of histidine phosphorylation.

Heterosis has been widely applied in watermelon breeding, because of the higher resistance and yield of hybrid. As the basis of heterosis utilization, genic male sterility (GMS) is an important tool for facilitating hybrid seed production, while the detailed mechanism in watermelon is still largely unknown. Here, we report a spontaneous mutant Se18 exhibited complete male sterility due to the uniquely multilayered tapetum and the un-meiotic pollen mother cells during pollen development. Using TMT based quantitative proteomic analyses, a total of 348 differentially abundant proteins (DAPs) were detected with the overwhelming majority down-regulated in mutant Se18. By analyzing the putative orthologs/homologs of Arabidopsis GMS related genes, the biosynthesis and transport of sporopollenin and tryphine precursors were predictably altered in mutant compared to its sibling wild type. Moreover, the general phenylpropanoid pathway as well as its related metabolisms was also expectably impaired in mutant, coincident with the pale yellow petals. Notably, some key transcriptional factors regulating tapetum development, together with their down-regulated targets, offered potentially valuable candidates regarding of male sterility. Collectively, the disrupted regulatory networks underlying male sterility of watermelon was proposed, which provide novel insights into genetic mechanism of male reproductive process and rich gene resources for future research. SIGNIFICANCE: Watermelon is an importantly economical cucurbit crop worldwide, with high nutritional value. Although several male sterile mutants have been identified in watermelon, the underlying molecular mechanism is poorly elucidated. Comparative cytological analysis revealed that the defective development of tapetum was responsible for male sterility in mutant Se18. Combined with the morphological comparison, male floral buds at 2.0-2.5 mm in diameter were confirmed with no obvious phenotypic differences but distinct cytological defects, which were in turn sampled for TMT based proteomic analyses. Referring to functionally characterized GMS related genes, the genetic pathway DYT1-TDF1-AMS-MS188-MS1 regulating tapetum development, together with some downstream targets, were considerably altered in mutant Se18. Moreover, enrichment analyses illustrated the general phenylpropanoid related metabolisms, as well as the biosynthesis and transport of sporopollenin and tryphine precursors, were significantly disrupted in defective anther development. Collectively, the proposed regulatory networks in watermelon not only contribute to a better understanding of molecular mechanisms underlying male sterility, but also provide valuable GMS related candidates for future researches.

Colorectal cancer (CRC) is a malignant tumour with high morbidity and mortality worldwide. Efficient screening strategies for CRC and pre-cancerous lesions can promote early medical intervention and treatment, thereby reducing morbidity and mortality. Proteins are generally considered key biomarkers of cancer. Herein, we performed a quantitative, original-tissue proteomics study in a cohort of ninety patients from pre-cancerous to cancerous conditions via liquid chromatography-tandem mass spectrometry. In total, 134,812 peptides, 8697 proteins, 2355 union differentially expressed proteins (DEPs), and 409 shared DEPs (compared with adjacent tissues) were identified. The number of DEPs indicated a positive correlation with increasing severity of illness. The union and shared DEPs were both enriched in the KEGG pathway of focal adhesion, metabolism of xenobiotics by cytochrome P450, and drug metabolism by cytochrome P450. Among the 2355 union DEPs, 32 were selected for identification and validation by multiple reaction monitoring from twenty plasma specimens. Of these, three proteins, transferrin receptor protein 1 (TFR1), adenosylhomocysteinase (SAHH), and immunoglobulin heavy variable 3-7 (HV307), were significantly differentially expressed and displayed the same expression pattern in plasma as observed in the tissue data. In conclusion, TFR1, SAHH, and HV307 may be considered as potential biomarkers for CRC screening. SIGNIFICANCE: Although CRC is a malignant tumour with high morbidity and mortality worldwide, efficient screening strategies for CRC and pre-cancerous lesions can play an important role in addressing these issues. Screening of molecular biomarkers provide a non-invasive, cost-effective, and efficient approach. Proteins are generally considered key molecular biomarkers of cancer. Our study reports a quantitative proteomics analysis of protein biomarkers for colorectal cancer (CRC) and adenomatous polyps, and identifies TFR1, SAHH, and HV307 as potential biomarkers for screening. This research makes a significant contribution to the literature as although mass spectrometry-based proteomics research has been widely used for clinical research, its application to clinical translation as parallel specimens ranging from pre-cancerous to cancerous tissues-according to the degree of disease progression-has not been readily assessed.

Erratum in J Proteomics. 2022 Mar 20;255:104465.

Quercus ilex is the dominant tree species in natural forest ecosystems across the Mediterranean Basin and in the agrosilvopastoral system dehesa, which has a high ecological and economical significance. As in other forestry species, survival in Q. ilex is threatened by long periods of drought. This paper reports the transcriptome and proteome profiles of 6-month-old seedlings subjected to severe drought conditions. Drought was imposed by water withholding in seedlings grown in perlite for 28 days. Seedling leaves were collected when leaf fluorescence had decreased by 20% and 45% relative to well-watered seedlings. The transcriptome and proteome were analyzed by using Illumina and shotgun platforms. The quality and confidence of the mRNA and protein identifications and quantifications were assessed, obtaining 25,169 transcripts and 3312 proteins. Variable transcripts and proteins were analyzed by Venn diagram, Pearson's correlation, GO enrichment, KEGG pathways, multivariate analysis and interaction networks. Despite the poor correlation between mRNA and protein, both platforms gave a complementary view of the changes in the abundance of several gene products under drought conditions and indicated that gene expression regulation and translation to phenotype is quite complex and gene-specific. As a general tendency, while transcripts and proteins of the metabolism were down-accumulated, those of stress related were up-accumulated. Out of the variable dataset, four gene products (viz., FtSH6, CLPB1, CLPB3, and HSP22) were up-accumulated at both omics levels at the two surveyed times, being the first work where they are described in drought response in forest species. These chaperones and proteases could be considered as potential drought tolerance markers to be used in the selection of elite, resilient genotypes, and in breeding programs.

Sclerotinia stem rot is a common disease found in Brassica rapa that is caused by the necrotic plant pathogen Sclerotinia sclerotiorum. Melatonin (MT) has known biological activity and effectively relieved this type of Sclerotinia stem rot in B. rapa. To better understand the mechanisms behind MT-induced S. sclerotiorum resistance in B. rapa, we performed both proteomic and metabolomic analysis. Our results showed that during S. sclerotiorum infection, thiamine synthesis was activated and defended against it. In infected leaves, ribosomal synthesis-related proteins responded positively to MT treatment. Integrated proteomic and metabolomic analysis showed that amino acid metabolism was activated by MT treatment. After MT treatment, adenosine-triphosphate (ATP) content and the activity of antioxidant enzymes were both increased in B. rapa infected leaves. Cysteine synthase, sulfur transfer-related proteins, and glucosinolate (GS) were all increased after MT treatment in infected B. rapa leaves. Taken together, these results indicated that B. rapa leaves promoted thiamine formation to defend against S. sclerotiorum infection. Moreover, MT helped further induce antioxidant activation in B. rapa in an ATP-dependent manner and stimulating GS biosynthesis to well inhibit the S. sclerotiorum infection. SIGNIFICANCE: Melatonin (MT) has biological activity and effectively relieved the Sclerotinia stem rot of Brassica rapa caused by the necrotic plant pathogen Sclerotinia sclerotiorum. In order to reveal the molecular mechanisms of MT-induced S. sclerotiorum resistance in B. rapa, comprehensive proteomic and metabolomic analyses were conducted. The integration analysis of omic-data illustrated that the modulation of ATP and glucosinolate biosynthesis induced by MT administration helped to defend the infection of S. sclerotiorum in B. rapa. Our results will provide insights into MT-induced anti-fungal mechanism and therapeutic strategies to mitigate Sclerotinia stem rot of B. rapa, thereby increasing plant yield and decreasing economic losses.

To investigate the mechanisms of the defense system and antioxidant defense system during chicken embryo development, protein profiling of liver tissues in chicken embryo at Day 16 and Day 20 was conducted. TMT was used to analyze the liver tissues proteomes with significantly different activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in chicken embryo. PRM was operated to validate the target differentially abundant proteins (DAPs) using the same samples. The result showed a total of 34 DAPs were identified. Among these, 9 were upregulated and 25 were downregulated. The screened DAPs strictly related to regulation of oxidoreductase activity (DDO and GAS2L1), response to stress (ERAD2 and SAA), immune system process (GAL3 and PDCD4), and lipid regulation and metabolism (ETNPPL, APOV1, LIPM, and APOA4). These analyses indicated that the antioxidant enzyme activity of chicken embryo is regulated through different pathways. Correlation analysis revealed a linear relationship between mRNA and protein expression and 12 genes (ORM1, C8B, KPNA2, CA4, C1S, SULT1B, ETNPPL, ERCC6L, DDO, SERPINF1, VAT1L, and APOA4) were detected to be differently expressed both at mRNA and protein levels. In consequence, these findings are an important resource that can be used in future studies of antioxidant mechanisms in chicken embryo. BIOLOGICAL SIGNIFICANCE: The genetic mechanisms of antioxidant activity are still unclear in chicken embryo. In the article, the combined transcriptomic and proteomic analysis is used to further explore potential signaling pathways and differentially abundant proteins related to antioxidant activity. These findings will facilitate a better understanding of the mechanism and these DAPs can be further investigated as candidate markers to predict the activity of antioxidant enzymes.