Comparative Proteomics and Phosphoproteomics Analysis Reveal the Possible Breed Difference in Yorkshire and Duroc Boar Spermatozoa

Sperm cells are of unique elongated structure and function, the development of which is tightly regulated by the existing proteins and the posttranslational modifications (PTM) of these proteins. Based on the phylogenetic relationships of various swine breeds, Yorkshire boar is believed to be distinctly different from Duroc boar. The comprehensive differential proteomics and phosphoproteomics profilings were performed on spermatozoa from both Yorkshire and Duroc boars. By both peptide and PTM peptide quantification followed by statistical analyses, 167 differentially expressed proteins were identified from 1,745 proteins, and 283 differentially expressed phosphopeptides corresponding to 102 unique differentially phosphorylated proteins were measured from 1,140 identified phosphopeptides derived from 363 phosphorylated proteins. The representative results were validated by Western blots. Pathway enrichment analyses revealed that majority of differential expression proteins and differential phosphorylation proteins were primarily concerned with spermatogenesis, male gamete generation, sperm motility, energy metabolism, cilium morphogenesis, axonemal dynein complex assembly, sperm–egg recognition, and capacitation. Remarkably, axonemal dynein complex assembly related proteins, such as SMCP, SUN5, ODF1, AKAP3, and AKAP4 that play a key regulatory role in the sperm physiological functions, were significantly higher in Duroc spermatozoa than that of Yorkshire. Furthermore, phosphorylation of sperm-specific proteins, such as CABYR, ROPN1, CALM1, PRKAR2A, and PRKAR1A, participates in regulation of the boar sperm motility mainly through the cAMP/PKA signal pathway in different breeds, demonstrating that protein phosphorylation may be an important mechanism underlying the sperm diversity. Protein–protein interaction analysis revealed that the 14 overlapped proteins between differential expression proteins and differential phosphorylation proteins potentially played a key role in sperm development and motility of the flagellum, including the proteins ODF1, SMCP, AKAP4, FSIP2, and SUN5. Taken together, these physiologically and functionally differentially expressed proteins (DEPs) and differentially expressed phosphorylated proteins (DPPs) may constitute the proteomic backgrounds between the two different boar breeds. The validation will be performed to delineate the roles of these PTM proteins as modulators of Yorkshire and Duroc boar spermatozoa.

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Identification of differentially expressed proteins in fresh and frozen–thawed boar spermatozoa by iTRAQ-coupled 2D LC–MS/MS

Reproduction ◽

10.1530/rep-13-0313 ◽

2014 ◽

Vol 147 (3) ◽

pp. 321-330 ◽

Cited By ~ 50

Author(s):

Xiaoli Chen ◽

Huabin Zhu ◽

Chuanhuo Hu ◽

Haisheng Hao ◽

Junfang Zhang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Bioinformatic Analysis ◽

Absolute Quantification ◽

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Protein Levels ◽

Boar Semen ◽

Isobaric Tags ◽

Boar Spermatozoa

Cryodamage is a major problem in semen cryopreservation, causing changes in the levels of proteins that influence the function and motility of spermatozoa. In this study, protein samples prepared from fresh and frozen–thawed boar spermatozoa were compared using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled to 2D LC–MS/MS analysis. A total of 41 differentially expressed proteins were identified and quantified, including 35 proteins that were present at higher levels and six proteins that were present at lower levels in frozen–thawed spermatozoa by at least a mean of 1.79-fold (P<0.05). On classifying into ten distinct categories using bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant to sperm premature capacitation, adhesions, energy supply, and sperm–oocyte binding and fusion. The expression of four of these proteins, SOD1, TPI1, ODF2, and AKAP3, was verified by western blot analysis. We propose that alterations in these identified proteins affect the quality of cryopreserved semen and ultimately lower its fertilizing capacity. This is the first study to compare protein levels in fresh and frozen–thawed spermatozoa using the iTRAQ technology. Our preliminary results provide an overview of the molecular mechanisms of cryodamage in frozen–thawed spermatozoa and theoretical guidance to improve the cryopreservation of boar semen.

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Improving Sperm Cryopreservation With Type III Antifreeze Protein: Proteomic Profiling of Cynomolgus Macaque (Macaca fascicularis) Sperm

Frontiers in Physiology ◽

10.3389/fphys.2021.719346 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bingbing Chen ◽

Shengnan Wang ◽

Briauna Marie Inglis ◽

Hao Ding ◽

Angbaji Suo ◽

...

Keyword(s):

Molecular Mechanism ◽

Sperm Motility ◽

Cynomolgus Macaque ◽

Antifreeze Protein ◽

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Proteomic Profiling ◽

Freezing And Thawing ◽

Proteomic Change ◽

Acrosomal Integrity

Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p < 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo–AFP) (p < 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 μg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.

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Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

PLoS ONE ◽

10.1371/journal.pone.0093947 ◽

2014 ◽

Vol 9 (4) ◽

pp. e93947 ◽

Cited By ~ 4

Author(s):

Fangyu Chen ◽

Liangrong Jiang ◽

Jingsheng Zheng ◽

Rongyu Huang ◽

Houcong Wang ◽

...

Keyword(s):

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Rice Seedlings ◽

Phosphorylated Proteins

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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

Virology Journal ◽

10.1186/s12985-021-01512-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Yuting Yang ◽

Yun Zhang ◽

Changcheng Yang ◽

Fang Fang ◽

Ying Wang ◽

...

Keyword(s):

Mass Spectrometry ◽

Avian Influenza ◽

Differential Expression ◽

Gel Electrophoresis ◽

A549 Cells ◽

Infected Group ◽

Differentially Expressed ◽

Mitochondrial Proteins ◽

Differentially Expressed Proteins ◽

Pathogenic Avian Influenza

Abstract Background Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. H5N1 viruses can cause severe damage and are associated with a high mortality rate, but H9N2 viruses do not cause such outcomes. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins related to H5N1 and H9N2 virus infections. Methods According to the determined viral infection titer, A549 cells were infected with 1 multiplicity of infection virus, and the mitochondria were extracted after 24 h of incubation. The protein from lysed mitochondria was analyzed by the BCA method to determine the protein concentration, as well as SDS-PAGE (preliminary analysis), two-dimensional gel electrophoresis, and mass spectrometry. Differential protein spots were selected, and Western blotting was performed to verify the proteomics results. The identified proteins were subjected to GO analysis for subcellular localization, KEGG analysis for functional classification and signaling pathways assessment, and STRING analysis for functional protein association network construction. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. Protein spots were further subjected to mass spectrometry identification and removal of redundancy, and 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The differential expression of 70-kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase was verified by Western blot, and the results were consistent with the proteomics findings. Functional analysis indicated that these differentially expressed proteins were primarily involved in apoptosis and metabolism. Conclusions Compared with their expression in the H9N2 group, the differential expression of eight mitochondrial proteins in the H5N1 group led to host T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2.

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In-Depth Proteomic Characterization of Lineage-Biased Hematopoietic Progenitor Cells in the Fetus and Adult

Blood ◽

10.1182/blood-2019-121799 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3703-3703

Author(s):

Maria Jassinskaja ◽

Kristýna Pimková ◽

Emil Johansson ◽

Ewa Sitnicka Quinn ◽

Jenny Hansson

Keyword(s):

Mass Spectrometry ◽

Progenitor Cells ◽

Differential Expression ◽

Normal Development ◽

Differentially Expressed ◽

Differential Sensitivity ◽

Hematopoietic Progenitor Cells ◽

Differentially Expressed Proteins ◽

Hematopoietic Progenitor ◽

The Difference

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and upon malignant transformation. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored at the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in hematopoietic stem and progenitor cells (HSPCs) during ontogeny regulates the outcome of hematopoiesis both during normal development and upon leukemia initiation, and that the proteomic makeup of the leukemia-initiating cell has an instructive role in determining the outcome of the resulting cancer. In our latest work, we utilized quantitative mass spectrometry-based proteomics to characterize and compare the proteomic makeup of fetal and adult Lin- Sca-1+ cKit+ (LSK) HSPCs (Jassinskaja et al., 2017, Cell Reports), representing all of the earliest stem and progenitors in fetal and adult hematopoiesis. We identified differences in several important cellular processes not previously described to play a role in hematopoiesis, highlighting the need for applying proteomic-centric approaches in the field. In order to further increase our understanding of normal and malignant hematopoiesis during ontogeny, we are now continuing this work by focusing on more stringently defined populations of lineage-biased hematopoietic progenitor cells (HPCs). Here, we have utilized encapsulated methods for preparation of microscale samples in combination with state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal (E14.5) and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-macrophage progenitors (GMPs). Our analysis resulted in the identification and quantification of 4189 proteins, with over 200 proteins per cell type displaying differential expression between the fetus and the adult. Importantly, the differentially expressed proteins were enriched for a broad variety of biological processes. Similar to our previous findings in HSPCs, for all three cell types, proteins higher expressed in the fetus showed a strong enrichment for cell cycle- and translation-related processes, whereas those higher expressed in the adult were enriched for processes related to immune response and redox homeostasis. Our preliminary analysis of hematopoietic cell subset signatures associated with the differentially expressed proteins suggests a stronger lymphoid bias in fetal compared to adult LMPPs as well as CLPs. Surprisingly, the proteomic signature of fetal GMPs suggests a retained megakaryocyte-erythroid potential, which is corroborated by a significantly higher expression of megakaryocyte progenitor marker CD41 on the fetal cells. Upon analyzing expression of transcription factors (TFs) in fetal and adult HPCs, we could confirm differential expression of TFs known to have ontogeny-specific roles in hematopoiesis (e.g. Arid3a and Etv6). Importantly, we also identified several differentially expressed TFs that could represent novel regulators of fetal- and adult-specific features of hematopoiesis, such as Irf8, Btf3, Mndal and Pura. Furthermore, the difference in expression of Irf8 observed here could indicate a previously unknown ontogenic switch in the balance between neutrophil and monocyte production from myeloid-competent progenitors. Lastly, our data shows strong indications of a differential sensitivity towards Rho kinase inhibition between the fetal and the adult HPCs. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults. Disclosures No relevant conflicts of interest to declare.

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Differential proteomics of tobacco seedling roots at high and low potassium concentrations

Scientific Reports ◽

10.1038/s41598-021-88689-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lin-jian Dai ◽

Yu-kun Liu ◽

Chong-wen Zhu ◽

Jun Zhong

Keyword(s):

Metabolic Pathways ◽

Acid Metabolism ◽

Tobacco Plant ◽

Large Subunit ◽

Protein Translation ◽

Protein Quantification ◽

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Differential Proteomics ◽

High Potassium

AbstractThe effects of high potassium and normal potassium treatments on protein expression in roots of flue-cured tobacco plant HKDN-5 at the seedling stage were analyzed by an unlabeled protein quantification technique. The results showed that 555 proteins were differentially expressed (245 proteins were down-regulated and 310 proteins were up-regulated) in high potassium treatment compared with normal potassium treatment. Differentially expressed proteins were involved in 96 metabolic pathways (42 metabolic pathways, 21 synthetic pathways as well as catabolic pathways, including fatty acid metabolism, phenylpropane biosynthesis, ketone body synthesis and degradation, and butyric acid metabolism. Root processing of high potassium concentrations leads to increases in the synthesis of peroxidase, superoxide dismutase and acyl-coenzyme-A synthetase. Additional proteomic differences observed in tobacco roots grown in high potassium include proteins involved with genetic information processing as well as environmental sensing. Examples include RNA helicase, ABC transporters and large subunit GTPases. These up-regulated differentially expressed proteins function mainly in protein translation, ribosome structure and protein synthesis. This indicates that under high potassium treatment, root protein synthetic processes are accelerated and substance metabolism pathways are enhanced; thus, providing the material and energetic basis for root growth.

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Label-Free Proteomic Analysis of Flavohemoglobin Deleted Strain of Saccharomyces cerevisiae

International Journal of Proteomics ◽

10.1155/2016/8302423 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12

Author(s):

Chiranjit Panja ◽

Rakesh K.S. Setty ◽

Gopal Vaidyanathan ◽

Sanjay Ghosh

Keyword(s):

Saccharomyces Cerevisiae ◽

Stress Responses ◽

Nitrosative Stress ◽

Nitrogen Compound ◽

Nuclear Gene ◽

Differentially Expressed ◽

Differentially Expressed Proteins ◽

Label Free ◽

Differential Proteomics ◽

Go Terms

Yeast flavohemoglobin, YHb, encoded by the nuclear gene YHB1, has been implicated in the nitrosative stress responses in Saccharomyces cerevisiae. It is still unclear how S. cerevisiae can withstand this NO level in the absence of flavohemoglobin. To better understand the physiological function of flavohemoglobin in yeast, in the present study a label-free differential proteomics study has been carried out in wild-type and YHB1 deleted strains of S. cerevisiae grown under fermentative conditions. From the analysis, 417 proteins in Y190 and 392 proteins in ΔYHB1 were identified with high confidence. Interestingly, among the differentially expressed identified proteins, 40 proteins were found to be downregulated whereas 41 were found to be upregulated in ΔYHB1 strain of S. cerevisiae (p value < 0.05). The differentially expressed proteins were also classified according to gene ontology (GO) terms. The most enriched and significant GO terms included nitrogen compound biosynthesis, amino acid biosynthesis, translational regulation, and protein folding. Interactions of differentially expressed proteins were generated using Search Tool for the Retrieval of Interacting Genes (STRING) database. This is the first report which offers a more complete view of the proteome changes in S. cerevisiae in the absence of flavohemoglobin.

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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

10.21203/rs.3.rs-60533/v1 ◽

2020 ◽

Author(s):

Yuting Yang ◽

Yun Zhang ◽

Changcheng Yang ◽

Fang Fang ◽

Ying Wang ◽

...

Keyword(s):

Avian Influenza ◽

Western Blot ◽

Differential Expression ◽

Gel Electrophoresis ◽

A549 Cells ◽

Infected Group ◽

Differentially Expressed ◽

Mitochondrial Proteins ◽

Differentially Expressed Proteins ◽

Pathogenic Avian Influenza

Abstract Background In the past 20–30 years, both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses could cause severe damage and a high death rate, but H9N2 viruses could not. In this study, we use H9N2 virus infection as a control to investigate the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections in A549 cells. Methods According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results, and then perform GO and KEGG analysis. Results In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Conclusions Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism and the cytoskeleton. The differential expression of eight mitochondrial proteins in H5N1-infected cells resulted in decreased T cell activation, decreased antigen presentation and stress response, reduced ATP synthesis, and decreased induction of apoptosis, resulting in the higher pathogenicity of H5N1 virus than H9N2 virus.These finding may provide a basis for analyzing the pathogenesis of influenza viruses with different virulence levels, identifying anti-influenza host targets and developing new influenza vaccines.

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Quantitative proteomics suggest a potential link between early embryonic death and trisomy 16

Reproduction Fertility and Development ◽

10.1071/rd17319 ◽

2019 ◽

Vol 31 (6) ◽

pp. 1116

Author(s):

Ting Yao ◽

Haiyan Hou ◽

Guozhong Liu ◽

Jun Wu ◽

Zhe Qin ◽

...

Keyword(s):

Differential Expression ◽

Quantitative Proteomics ◽

Transforming Growth Factor ◽

Inflammatory Responses ◽

Reference Group ◽

Differentially Expressed ◽

Matrix Metalloproteinase 2 ◽

Differentially Expressed Proteins ◽

Trisomy 16 ◽

Embryonic Death

Activation of extracellular signal-regulated kinase (ERK) signalling, alteration of the uterine microenvironment and a reduction in human chorionic gonadotrophin production have been linked with fetal trisomy 16-induced early embryonic death (EED). However, the detailed biological mechanism of EED remains unclear. Using quantitative proteomics we successfully screened differentially expressed proteins in the villous tissues from patients with EED and fetal trisomy 16 (EEDT16), patients with EED but normal fetal chromosomes (EEDNC) and patients undergoing elective abortion with normal fetal chromosomes (EANC) as the reference group. Compared with the reference group, we identified 337 and 220 differentially expressed proteins in EEDT16 patients and EEDNC patients respectively; these were involved in critical biological processes including immune response, superoxide metabolism, inflammatory responses and so on. We found that differential expression of immunological function-related molecules, such as human leukocyte antigen-g (HLA-G), HLA-C, Fc Fragment Of IgG Receptor III (FcγR III), also named CD16, interleukin 18 (IL-18) and transforming growth factor β1 (TGF-β1), might induce EED in both EEDT16 and EEDNC patients. More severe immunological dysfunction was observed in EEDT16 patients than that in EEDNC patients. Furthermore, differential expression of implantation and invasion-related molecules, such as cytochrome b-245 light chain (CYBA), neutrophil cytosol factor 2 (NCF2), Mitogen-activated protein kinase kinase kinase 4 (MAP3K4), matrix metalloproteinase 2 (MMP2), MMP9 and tumour necrosis factor α (TNF-α) might induce EED in both EEDT16 and EEDNC patients, although more severe dysfunction in the implantation and invasion ability of villous tissues was observed in EEDT16 patients.

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Differential mitochondrial proteomic analysis of A549 cells infected with avian influenza virus subtypes H5 and H9

10.21203/rs.3.rs-60533/v2 ◽

2020 ◽

Author(s):

Yuting Yang ◽

Yun Zhang ◽

Changcheng Yang ◽

Fang Fang ◽

Ying Wang ◽

...

Keyword(s):

Avian Influenza ◽

Western Blot ◽

Differential Expression ◽

Gel Electrophoresis ◽

A549 Cells ◽

Infected Group ◽

Differentially Expressed ◽

Mitochondrial Proteins ◽

Differentially Expressed Proteins ◽

Pathogenic Avian Influenza

Abstract Background: Both the highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have been reported to cross species barriers to infect humans. However, H5N1 viruses can cause severe damage and a high mortality rate, but H9N2 viruses can not. Our purpose was to use proteomics technology to study the differential expression of mitochondrial-related proteins caused by H5N1 and H9N2 virus infections.Methods: According to the determined viral infection titer, A549 cells were infected with 1 MOI (multiplicity of infection) virus, and the mitochondria were extracted after 24 hours of incubation. The lysed mitochondrial protein was analyzed by BCA method for protein concentration, SDS-PAGE preliminary analysis, two-dimensional gel electrophoresis, and mass spectrometry. Select different protein spots, perform Western Blot to verify the proteomics results. The identified proteins use GO analysis for subcellular localization, KEGG analysis for functional classification and signal pathways, STRING analysis for functional protein association networks.Results: In the 2-D gel electrophoresis analysis, 227 protein spots were detected in the H5N1-infected group, and 169 protein spots were detected in the H9N2-infected group. After further MS identification and removal of redundancy, 32 differentially expressed proteins were identified. Compared with the H9N2 group, the H5N1-infected group had 16 upregulated mitochondrial proteins and 16 downregulated proteins. The 70 kDa heat shock protein analogs, short-chain enoyl-CoA hydratase, malate dehydrogenase, and ATP synthase were verified by Western Blot, and the results were consistent with proteomics. Functional analysis indicated that these differentially expressed proteins were involved mainly in apoptosis, metabolism.Conclusions: Compared with H9N2 group, the differential expression of eight mitochondrial proteins in H5N1 group led to T cell activation, antigen presentation, stress response, ATP synthesis and cell apoptosis reduction, leading to higher pathogenicity of H5N1 than H9N2 virus.

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