Label-free Quantitative Proteomic Approach Research Articles

Reversible SAHH inhibitor protects against glomerulonephritis in lupus-prone mice by downregulating renal \u03b1-actinin-4 expression and stabilizing integrin-cytoskeleton linkage

BackgroundGlomerulonephritis is one of the major complications and causes of death in systemic lupus erythematosus (SLE) and is characterized by glomerulosclerosis, interstitial fibrosis, and tubular atrophy, along with severe persistent proteinuria. DZ2002 is a reversible S-adenosyl-l-homocysteine hydrolase (SAHH) inhibitor with potent therapeutic activity against lupus nephritis in mice. However, the molecular events underlying the renal protective effects of DZ2002 remained unclear. This study is designed to uncover the molecular mechanisms of DZ2002 on glomerulonephritis of lupus-prone mice.MethodsWe conducted a twice-daily treatment of DZ2002 on the lupus-prone NZB/WF1 mice, and the progression of lupus nephritis and alteration of renal function were monitored. The LC-MS-based label-free quantitative (LFQ) proteomic approach was applied to analyze the kidney tissue samples from the normal C57BL/6 mice and the NZB/WF1 mice treated with DZ2002 or vehicle. KEGG pathway enrichment and direct protein-protein interaction (PPI) network analyses were used to map the pathways in which the significantly changed proteins (SCPs) are involved. The selected proteins from proteomic analysis were validated by Western blot analysis and immunohistochemistry in the kidney tissues.ResultsThe twice-daily regimen of DZ2002 administration significantly ameliorated the lupus nephritis and improved the renal function in NZB/WF1 mice. A total of 3275 proteins were quantified, of which 253 proteins were significantly changed across normal C57BL/6 mice and the NZB/WF1 mice treated with DZ2002 or vehicle. Pathway analysis revealed that 13 SCPs were involved in tight junction and focal adhesion process. Further protein expression validation demonstrated that DZ2002-treated NZB/WF1 mice exhibited downregulation of α-actinin-4 and integrin-linked kinase (ILK), as well as the restoration of β1-integrin activation in the kidney tissues compared with the vehicle-treated ones.ConclusionsOur study demonstrated the first evidence for the molecular mechanism of SAHH inhibitor on glomerulonephritis in SLE via the modulation of α-actinin-4 expression and focal adhesion-associated signaling proteins in the kidney.

Lipid surface modifications increase mesoporous silica nanoparticle labeling properties in mesenchymal stem cells.

BackgroundNanoparticles have emerged as promising cell-labeling tools, as they can be precisely tailored in terms of chemical and physical properties. Mesoporous silica nanoparticles (MSNs), in particular, are easily tunable with regard to surface and core chemistry, and are able to confine dyes and drug molecules efficiently.PurposeThe aim of this study was to investigate the effect of lipid and polyethylene glycol (PEG) surface modifications on MSN stem-cell-tracking abilities.MethodsLipid and PEG surface functionalized MSNs were synthesized and the effect of surface functionalization on cell internalization, proliferation, differentiation and cell proteomics was investigated in patient derived mesenchymal stem cells (MSCs).ResultsMSNs and lipid surface-modified MSNs were internalized by >80% of the MSC population, with the exception of nanoparticles modified with short PEG chains (molecular weight 750 [MSN-PEG750]). Lipid-modified MSNs had higher labeling efficiency with maximum uptake after 2 hours of exposure and were in addition internalized 17 times higher compared to unmodified MSNs, without negatively affecting differentiation capacity. Using a mass-spectrometry-based label-free quantitative proteomics approach, we show that MSN labeling leads to the up- and downregulation of proteins that were unique for the different surface-modified MSNs. In addition, functional enrichments were found in human MSCs labeled with MSNs, MSN-PEG750, and lipid-modified MSNs.SummaryHere we show that organic modifications with lipids and PEGylation can be used as a promising strategy to improve MSN labeling capabilities. In particular, we show that lipid modifications can optimize such probes in three distinct ways: significantly improved signal strength, a barrier for sustained release of additional probes, and improved stem-cell-labeling efficiency.

The proteomic study of serially passaged human skin fibroblast cells uncovers down-regulation of the chromosome condensin complex proteins involved in replicative senescence

Dermal fibroblast is one of the major constitutive cells of skin and plays a central role in skin senescence. The replicative senescence of fibroblasts may cause skin aging, bad wound healing, skin diseases and even cancer. In this study, a label-free quantitative proteomic approach was employed to analyzing the serial passaged human skin fibroblast (CCD-1079Sk) cells, resulting in 3371 proteins identified. Of which, 280 proteins were significantly changed in early passage (6 passages, P6), middle passage (12 passages, P12) and late passage (21 passages, P21), with a time-dependent decrease or increase tendency. Bioinformatic analysis demonstrated that the chromosome condensin complex, including structural maintenance of chromosomes protein 2 (SMC2) and structural maintenance of chromosomes protein 4 (SMC4), were down-regulated in the serially passaged fibroblast cells. The qRT-PCR and Western Blot experiments confirmed that the expression of these two proteins were significantly down-regulated in a time-dependent manner in the subculture of human skin fibroblasts (HSFb cells). In summary, we used serially passaged human skin fibroblast cells coupled with quantitative proteomic approach to profile the protein expression pattern in the temporal progress of replicative senescence in HSFb cells and revealed that the down-regulation of the chromosome condensin complex subunits, such as SMC2 and SMC4, may play an important role in the fibroblast senescence.

Deep proteomic network analysis of Alzheimer\u2019s disease brain reveals alterations in RNA binding proteins and RNA splicing associated with disease

BackgroundThe complicated cellular and biochemical changes that occur in brain during Alzheimer’s disease are poorly understood. In a previous study we used an unbiased label-free quantitative mass spectrometry-based proteomic approach to analyze these changes at a systems level in post-mortem cortical tissue from patients with Alzheimer’s disease (AD), asymptomatic Alzheimer’s disease (AsymAD), and controls. We found modules of co-expressed proteins that correlated with AD phenotypes, some of which were enriched in proteins identified as risk factors for AD by genetic studies.MethodsThe amount of information that can be obtained from such systems-level proteomic analyses is critically dependent upon the number of proteins that can be quantified across a cohort. We report here a new proteomic systems-level analysis of AD brain based on 6,533 proteins measured across AD, AsymAD, and controls using an analysis pipeline consisting of isobaric tandem mass tag (TMT) mass spectrometry and offline prefractionation.ResultsOur new TMT pipeline allowed us to more than double the depth of brain proteome coverage. This increased depth of coverage greatly expanded the brain protein network to reveal new protein modules that correlated with disease and were unrelated to those identified in our previous network. Differential protein abundance analysis identified 350 proteins that had altered levels between AsymAD and AD not caused by changes in specific cell type abundance, potentially reflecting biochemical changes that are associated with cognitive decline in AD. RNA binding proteins emerged as a class of proteins altered between AsymAD and AD, and were enriched in network modules that correlated with AD pathology. We developed a proteogenomic approach to investigate RNA splicing events that may be altered by RNA binding protein changes in AD. The increased proteome depth afforded by our TMT pipeline allowed us to identify and quantify a large number of alternatively spliced protein isoforms in brain, including AD risk factors such as BIN1, PICALM, PTK2B, and FERMT2. Many of the new AD protein network modules were enriched in alternatively spliced proteins and correlated with molecular markers of AD pathology and cognition.ConclusionsFurther analysis of the AD brain proteome will continue to yield new insights into the biological basis of AD.

Proteomics analysis in A375 melanoma cells before and after short hairpin RNA-mediated Cbl-b gene silencing

Objective To analyze differentially expressed proteins in A375 melanoma cells before and after short hairpin RNA (shRNA) -mediated Cbl-b gene silencing. Methods The label-free quantitative proteomics approach was performed to identify differentially expressed proteins between A375 cells transfected with lentiviral vectors containing Cbl-b shRNA (Cbl-b shRNA group) and those with control lentiviral vectors (control group) . Then, the properties of differentially expressed proteins were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis. Western blot analysis was conducted to determine the expression of differential proteins (EphA2 and GSK3β) and phosphorylated protein kinase (p-AKT) after shRNA-mediated Cbl-b gene silencing. Statistical analysis was carried out by t test of two independent-samples for comparison of protein expression abundance between the two groups with SPSS 23.0 software. Additionally, the results of GO and KEGG enrichment analysis were analyzed by Fisher′s exact test. Results A total of 3 449 proteins were identified and quantified, and 74 of them were differentially expressed between the Cbl-b shRNA group and control group. Compared with the control group, 52 proteins were up-regulated and 22 were down-regulated in the Cbl-b shRNA group. GO enrichment analysis of differential proteins revealed that the top five significantly enriched biological processes were integrin-mediated cell adhesion, single-organism metabolic process, regulation of integrin-mediated cell adhesion, regulation of protein-targeting mitochondria and nucleic acid metabolic process. The top five significantly enriched molecular functions included DNA binding, 2-iron, 2-sulfur cluster binding, signaling receptor activity, cadherin binding and cell adhesion molecule binding. The top five significantly enriched cell components included nucleosome, DNA packaging complex, photoreceptor connecting cilium, DNA-protein complex and extracellular region part. KEGG enrichment analysis demonstrated that the top five significantly enriched melanoma-related signaling pathways were folate biosynthesis, axon guidance, extracellular matrix-receptor interaction, adherens junction and Wnt signaling pathways. As Western blot analysis revealed, the Cbl-b shRNA group showed lower protein expression of EphA2 (0.369) , but higher protein expression of GSK3β (3.524) compared with the control group (1) , which were consistent with the results of proteomics analysis. Additionally, the protein expression of p-AKT was down-regulated in Cbl-b shRNA group (0.453) compared with the control group (1) . Conclusion Cbl-b may be involved in the occurrence of melanoma through a variety of biological pathways, and the EphA2/PI3K/AKT signaling pathway may be one important pathway. Key words: Melanoma; Ubiquitin-protein ligases; Proto-oncogene proteins cbl-b; RNA, small interfering; Proteomics

Proteomics Unravels the Regulatory Mechanisms in Human Tears Following Acute Renouncement of Contact Lens Use: A Comparison between Hard and Soft Lenses

Contact lenses (CLs) provide a superior alternative to spectacles. Although beneficial, the global burden of ocular dysfunctions attributed to regular use of CLs remains a topic of much challenge in ophthalmic research owing to debilitating clinical repercussions on the ocular surface, which are often manifested as breach in tear film integrity. This study elucidated the intricate tear proteome changes attributed to the use of different CLs (hard and soft) and unravelled, for the first time, the restorative mechanisms of several protein clusters following acute renouncement of CL use employing the label-free mass spectrometry-based quantitative proteomics approach. The expression patterns of certain proteins clusters were specific to the use of a particular lens type and a large majority of these actively regulates cell death and survival and, modulates cellular movement on the ocular surface. Noteworthy, CL use also evoked a significant upregulation of glycolytic enzymes associated with hypoxia and corresponding cognate metabolic pathways, particularly glucose metabolism and FXR/RXR pathways. Importantly, the assessment of CL renouncement unravelled the restorative properties of several clusters of proteins involved mainly in organismal injury and abnormalities and, cellular function and maintenance. These proteins play key roles in restoring tear homeostasis and wound-healing mechanisms post-CL use-elicited injury.

Mechanism of reduced insecticidal protein expression in Bt cotton under high temperature and drought based on proteomics.

To provide theoretical basis for the safety of insecticidal efficiency in Bt cotton, the effects of high temperature and drought stress on insecticidal protein expression and protein diffe-rently expression profile was studied. In this study, the Bt cotton cultivar Sikang 3 was used as expe-rimental material, with two treatments (40% field capacity and 38 ℃, HD, and 60% field capacity and 32 ℃, CK). Differences in proteomics of Bt cotton between HD and CK were compared using label-free quantitative proteomics technology. The results showed that high temperature and drought caused a significant reduction of insecticidal protein content in bolls, with a decrease of 38.2 ng·g-1 FM. The analysis of differential protein expression by label-free quantitative proteomic approach showed that 83 proteins were significantly up-regulated, but 104 proteins were significantly down-regulated in HD stressed cotton plants compared with CK. 122 new proteins were detected and 167 proteins expression was not observed under stressed conditions. Results from the enrichment analysis of differently expressed protein between two treatments showed that 14 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were affected under stress. Three KEGG pathways were related to the Bt protein synthesis and degradation: carbohydrate digestion and absorption pathway, protein export pathway, and protein processing in endoplasmic reticulum pathway. In the carbohydrate digestion and absorption pathway, the starch hydrolysis ability of HD treated cotton plants increased, while the ability to phosphorylate the hexoses, fructose and glucose decreased. In protein export pathway, the peptide synthesis in HD treatment was not significantly affected, while the process of transferring peptides into the endoplasmic reticulum was prohibited. In the protein processing in endoplasmic reticulum pathway, the ability of ubiquitin mediated proteolysis was increased in HD treatment.

Comparative proteomic analysis of mouse models of pathological and physiological cardiac hypertrophy, with selection of biomarkers of pathological hypertrophy by integrative proteogenomics

To determine fundamental characteristics of pathological cardiac hypertrophy, protein expression profiles in two widely accepted models of cardiac hypertrophy (swimming-trained mouse for physiological hypertrophy and pressure-overload-induced mouse for pathological hypertrophy) were compared using a label-free quantitative proteomics approach. Among 3955 proteins (19,235 peptides, false-discovery rate < 0.01) identified in these models, 486 were differentially expressed with a log2 fold difference ≥ 0.58, or were detected in only one hypertrophy model (each protein from 4 technical replicates, p < .05). Analysis of gene ontology biological processes and KEGG pathways identified cellular processes enriched in one or both hypertrophy models. Processes unique to pathological hypertrophy were compared with processes previously identified in cardiac-hypertrophy models. Individual proteins with differential expression in processes unique to pathological hypertrophy were further confirmed using the results of previous targeted functional analysis studies. Using a proteogenomic approach combining transcriptomic and proteomic analyses, similar patterns of differential expression were observed for 23 proteins and corresponding genes associated with pathological hypertrophy. A total of 11 proteins were selected as early-stage pathological-hypertrophy biomarker candidates, and the results of western blotting for five of these proteins in independent samples confirmed the patterns of differential expression in mouse models of pathological and physiological cardiac hypertrophy.

Abstract 164: Ston2 knockdown promotes stem-like properties via dnmt1-mediated muc1 upregulation in ovarian cancer

Abstract Cancer stem cells possess the ability of self-renewal, unlimited proliferation,and non-sensitivity to physical and chemical factors, which is regarded to be a cause of ovarian cancer recurrence, but the mechanism is currently unclear. We found that the expression of STON2 was significantly lower in ovarian cancer-stem like cells by LC-MS/MS-based label free quantitative proteomics approach and western blot validation. STON2 knockdown upregulated the expression of the CSC-related markers in protein level, increased the spheroids number and CD44+CD24−proportion. Besides, in vivo xenograft experiments, the volume of tumors from the shSton2 group were much bigger compared with the shNC group. Moreover, STON2 overexpression decreased the CSC-related markers and inhibited the spheroids number compared with the control group. Transcriptome sequencing data showed that MUC1 is negative correlation with STON2, which were verificated by RT-PCR and western blot analysis. Subsequently, we found that MUC1 mediated the role of STON2 in regulation of stem-like properties in ovarian cancer. MUC1 knockdown resulted in a significant decrease in the sphere-forming ability, CSC-related markers and CD44+CD24−proportion. In addition, MUC1 overexpression caused an increment of CSC-related markers expression and CD44+CD24−proportion. Furthermore, we found that the reducing of sphere-forming ability caused by STON2 overexpression was rescued by MUC1 overexpression. As MUC1 can be regulated through epigenetic mechanisms, we found that DNA methylation inhibitor Azacitidine was able to rescue the upregulation of MUC1 by STON2. The DNA methylation levels of MUC1 in one CpG-rich region were then evaluated with pyrosequencing, and the data displayed that the siSton2 group is lower than the control group. Furthermore, we found that the DNMT1 as a member of the DNA methyltransferase family decreased following with STON2 knockdown in protein level, rather than in mRNA level. We thus knock down DNMT1 using specific siRNA, and found that MUC1 overexpression both in mRNA level and protein level. In this study, we focused primarily on STON2 repression leading to DNMT1 downregulation and MUC1 activation is a main mechanism underlying the maintenance of stem-like properties in ovarian cancer. Citation Format: Shanshan Xu. Ston2 knockdown promotes stem-like properties via dnmt1-mediated muc1 upregulation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 164.

A proteomic insight into the MSP1 and flg22 induced signaling in Oryza sativa leaves

Previously, we reported a novel Magnaporthe oryzae- secreted protein MSP1, which triggers cell death and pathogen-associated molecular pattern (PAMP)-triggered immune (PTI) responses in rice. To investigate the MSP1 induced defense response in rice at the protein level, we employed a label-free quantitative proteomic approach, in parallel with flg22 treatment, which is a well-known elicitor. Exogenous application of MSP1 to rice leaves induced an oxidative burst, MAPK3/6 activation, and activation of pathogenesis-related genes (DUF26, PBZ, and PR-10). MaxQuant based label free proteome analysis led to the identification of 4167 protein groups of which 433 showed significant differences in response to MSP1 and/or flg22 treatment. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins associated mainly with the stress response, post-translational modification and signaling were increased in abundance. Moreover, several peroxidases and receptor kinases were induced by both the elicitors, highlighting their involvement in MSP1 and flg22 induced signaling in rice. Taken together, the results reported here contribute to our understanding of MSP1 and flg22 triggered immune responses at the proteome level, thereby increasing our overall understanding of PTI signaling in rice. Biological significanceMSP1 is a M. oryzae secreted protein, which triggers defense responses in rice. Previous reports have shown that MSP1 is required for the pathogenicity of rice blast fungus, however, the exact mechanism of its action and its downstream targets in rice are currently unknown. Identification of the downstream targets is required in order to understand the MSP1 induced signaling in rice. Moreover, key proteins identified could also serve as potential candidates for the generation of disease resistance crops by modulating stress signaling pathways. Therefore, here we employed, for the first time, a label-free quantitative proteomic approach to investigate the MSP1 induced signaling in rice together with flg22. Functional annotation of the differential proteins showed that majority of the proteins related to primary, secondary, and lipid metabolism were decreased, while proteins related to the defense response, signaling and ROS detoxification were majorly increased. Thus, as an elicitor, recombinant MSP1 proteins could be utilized to inducing broad pathogen resistance in crops by priming the local immune responses.

Proteomic study of endothelial dysfunction induced by AGEs and its possible role in diabetic cardiovascular complications

Endothelial dysfunction is one of the primary steps in the development of diabetes associated cardiovascular diseases. Hyperglycemic condition in diabetes promotes accumulation of advanced glycation end products (AGEs) in the plasma, that interact with the receptor for AGEs (RAGE) present on the endothelial cells and negatively affect their function. Using Human umbilical vascular endothelial cells (HUVECs) in culture, the effect of glycated human serum albumin on global proteomic changes was studied by SWATH-MS, a label free quantitative proteomic approach. Out of the 1860 proteins identified, 161 showed higher abundance while 123 showed lesser abundance in cells treated with glycated HSA. Bioinformatic analysis revealed that the differentially regulated proteins were involved in various processes such as apoptosis, oxidative stress etc. that are associated with endothelial dysfunction. Furthermore, the iRegulon analysis and immunofuorescence studies indicated that several of the differentially regulated proteins were transcriptionally regulated by NF-κB, that is downstream to AGE-RAGE axis. Some of the important differentially regulated proteins include ICAM1, vWF, PAI-1 that affect important endothelial functions like cell adhesion and blood coagulation. qPCR analysis showed an increase in expression of the AGE receptor RAGE along with other genes involved in endothelial function. AGE treatment to HUVEC cells led to increased oxidative stress and apoptosis. This is the first proteomics study that provides insight into proteomic changes downstream to AGE-RAGE axis leading to endothelial dysfunction and predisposing to cardiovascular complications. SignificanceCardiovascular disease (CVD) is a major pathological outcome in diabetic patients and it is important to address ways that target its development before the onset. Elevated plasma AGEs in diabetes can affect endothelial function and can continue to show their effects even after blood glucose levels are back to normal. Since endothelial dysfunction acts as one of the initiating factors for the development of CVD, understanding how AGEs affect the endothelial cell proteome to cause dysfunction will provide insight into the mechanisms involved and aid designing new therapeutic approaches.

Genomic and proteomic analysis of lignin degrading and polyhydroxyalkanoate accumulating \u03b2-proteobacterium Pandoraea sp. ISTKB

BackgroundLignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysaccharide-based biorefinery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to find the important enzymes for polymer utilization.ResultsGenomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profile on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase–peroxidase for lignin degradation. A strong stress response and detoxification mechanism was discovered. The two important gene clusters for lignin degradation and three PHA polymerase spanning gene clusters were identified and all the clusters were functionally active on KL–VA.ConclusionsThe unusual aerobic ‘-CoA’-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4–5% of total sequenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chemistry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profile. Genomic and proteomic approaches provided validation to this strain’s robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future.

Prediction of skin anti-aging clinical benefits of an association of ingredients from marine and maritime origins: Ex vivo evaluation using a label-free quantitative proteomic and customized data processing approach.

The application of ingredients from marine and maritime origins is increasingly common in skin care products, driven by consumer expectations for natural ingredients. However, these ingredients are typically studied for a few isolated invitro activities. The purpose of this study was to carry out a comprehensive evaluation of the activity on the skin of an association of ingredients from marine and maritime origins using label-free quantitative proteomic analysis, in order to predict the clinical benefits if used in a skin care product. An aqueous gel containing 6.1% of ingredients from marine and maritime origins (amino acid-enriched giant kelp extract, trace element-enriched seawater, dedifferentiated sea fennel cells) was topically applied on human skin explants. The skin explants' proteome was analyzed in a label-free manner by high-performance liquid nano-chromatography coupled with tandem mass spectrometry. A specific data processing pipeline (CORAVALID) providing an objective and comprehensive interpretation of the statistically relevant biological activities processed the results. Compared to untreated skin explants, 64 proteins were significantly regulated by the gel treatment (q-value≤0.05). Computer data processing revealed an activity of the ingredients on the epidermis and the dermis. These significantly regulated proteins are involved in gene expression, cell survival and metabolism, inflammatory processes, dermal extracellular matrix synthesis, melanogenesis and keratinocyte proliferation, migration, and differentiation. These results suggest that the tested ingredients could help to preserve a healthy epidermis and dermis, and possibly to prevent the visible signs of skin aging.

Comparative proteome analysis of the capsule from patients with frozen shoulder

The etiology of frozen shoulder (FS) is unclear. Accordingly, this study used a label-free quantitative shotgun proteomic approach to elucidate the pathogenesis of FS based on protein expression levels. Tissue samples from the rotator interval (RI), middle glenohumeral ligament (MGHL), and anterior-inferior glenohumeral ligament (IGHL) were collected from 12 FSs with severe stiffness and 7 shoulders with a rotator cuff tear (RCT) as controls. Protein mixtures were digested and analyzed by nano-liquid chromatography/electrospray ionization-tandem mass spectrometry. Relative protein expression levels were calculated by the signal intensity of identified peptide ions on mass spectra. Differentially expressed proteins between FS and RCT samples were evaluated by a gene enrichment analysis using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. We identified 1594 proteins, 1358 of which were expressed in all 6 tissue groups. We detected more upregulated proteins in the upper (RI and MGHL) FS groups and the lower (IGHL) RCT group than in the comparative groups, respectively. Various proteins with functions in tissue repair, collagen metabolism and fibrillation, cell-cell and cell-matrix adhesion, blood coagulation, and the immune response were expressed more highly in the RI and MGHL FS groups than in the RCT group. Proteins with functions in phagocytosis, glutathione metabolism, retinoid metabolism, and cholesterol metabolism were expressed more highly in the IGHL RCT group than in the FS group. The pathophysiology of FS differs between the upper and lower parts of the joint capsule. Different treatment strategies for FS may be appropriate, depending on the location.

Label free-based proteomic analysis of proteins in Bacillus cereus spores regulated by high pressure processing and slightly acidic electrolyzed water treatment

To investigate the mechanism of high pressure processing (HPP, 200 MPa/20 min + 500 MPa/20 min) and slightly acidic electrolyzed water (SAEW, 44 mg/L available chlorine concentration (ACC)) on B. cereus spore inactivation, a label-free quantitative proteomics approach based on high-resolution mass spectrometry (MS) data was used. Quantification of 2810 proteins was obtained with high confidence. A total of 93 and 83 proteins showed significant differential abundance after HPP-SAEW and HPP treatments, respectively, and 21 proteins changed significantly when both treatments were compared. The results indicated that the spore cortex-lytic enzyme was upregulated 3.15 fold after HPP treatment, and the germination was influenced by ATP generation. The metabolic, degradation, signaling, and biosynthesis pathways were involved in HPP-SAEW mediated spore inactivation. Amino acid-biosynthetic and quorum sensing were the main KEGG pathways mediated by SAEW under HPP conditions. Specifically, our results showed that the phosphorylation of the Spo0A transcription factor was mediated by the downregulation of the ABC transporter ATP-binding protein, which was associated with inactivation of spores.

Longissimus dorsi muscle label-free quantitative proteomic reveals biological mechanisms associated with intramuscular fat deposition

The pathways involved in intramuscular fat (IMF) deposition in Longissimus dorsi muscle were investigated using an integrated transcriptome-assisted label-free quantitative proteomic approach by High Definition Mass Spectrometry. We quantified 1582 proteins, of which 164 were differentially abundant proteins (DAPs, p < 0.05) between animals with high (H) and low (L) genomic estimated breeding values (GEBV) for IMF content. Ingenuity pathway analysis (IPA) revealed that these DAPs were mainly involved in glycolysis metabolism, actin cytoskeleton signaling, cell-cell adherens junction and pathways for MAPK and insulin. A comparative study between transcriptomic (mRNA) and proteomic data showed 17 differentially expressed genes corresponding to DAPs, of which three genes/proteins did not agree on the direction of the fold change between groups. Moreover, we investigated microRNAs data to explain these differences in fold change direction, being able to unravel two of the three unexpected mRNA/protein relationships. Results demonstrated that changes in protein/mRNA levels of sarcomere organization, intracellular signal transduction and regulation of actin cytoskeleton, are involved in IMF deposition. These findings provide a deeper understanding of the highly complex regulatory mechanisms involved in IMF deposition in cattle and indicate target pathways for future studies. SignificanceIntramuscular fat is the amount of fat deposited inside muscle and plays an important role in human health and meat quality attributes, influencing energy metabolism of skeletal muscle, as well as, tenderness, flavor, and juiciness of beef. We performed for the first time the utilization of integrated transcriptome-assisted label-free quantitative proteomic approach using High Definition Mass Spectrometry for characterization of the changes in the proteomic profile of the Longissimus dorsi muscle associated with intramuscular fat deposition in cattle. Furthermore, we compared the muscle proteome with the muscle transcriptome (mRNA and microRNAs), obtained by RNA-sequencing, to better understand the relationship between expression of mRNAs and proteins and to unravel essential biological mechanisms involved in bovine skeletal muscle IMF deposition.

Disentangling thermal stress responses in a reef-calcifier and its photosymbionts by shotgun proteomics

The proliferation of key marine ecological engineers and carbonate producers often relies on their association with photosymbiotic algae. Evaluating stress responses of these organisms is important to predict their fate under future climate projections. Physiological approaches are limited in their ability to resolve the involved molecular mechanisms and attribute stress effects to the host or symbiont, while probing and partitioning of proteins cannot be applied in organisms where the host and symbiont are small and cannot be physically separated. Here we apply a label-free quantitative proteomics approach to detect changes of proteome composition in the diatom-bearing benthic foraminifera Amphistegina gibbosa experimentally exposed to three thermal-stress scenarios. We developed a workflow for protein extraction from less than ten specimens and simultaneously analysed host and symbiont proteomes. Despite little genomic data for the host, 1,618 proteins could be partially assembled and assigned. The proteomes revealed identical pattern of stress response among stress scenarios as that indicated by physiological measurements, but allowed identification of compartment-specific stress reactions. In the symbiont, stress-response and proteolysis-related proteins were up regulated while photosynthesis-related proteins declined. In contrast, host homeostasis was maintained through chaperone up-regulation associated with elevated proteosynthesis and proteolysis, and the host metabolism shifted to heterotrophy.

Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis.

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses.

Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic balance under stress. In conclusion, our results provide a better understanding of the global metabolic adaptations of B. abortus associated with distinct environmental stresses. The identification of proteins necessary for stress resistance is crucial toward elucidating the infectious process in order to control brucellosis, and may facilitate the discovery of novel therapeutic targets and effective vaccines.

Quantitative proteomic Analysis Reveals up-regulation of caveolin-1 in FOXP3-overexpressed human gastric cancer cells

Forkhead box protein 3 (FOXP3) is implicated in tumor progression and prognosis in various types of tumor cells. We have recently reported that FOXP3 inhibited proliferation of gastric cancer (GC) cells through activating the apoptotic signaling pathway. In this study, we found that over-expression of FOXP3 inhibited GC cell migration, invasion and proliferation. Then, the label-free quantitative proteomic approach was employed to further investigating the down-stream proteins regulated by FOXP3, resulting in a total of 3,978 proteins quantified, including 186 significantly changed proteins. Caveolin-1 (CAV1), as a main constituent protein of caveolae, was one of those changed proteins up-regulated in FOXP3-overexpressed GC cells, moreover, it was assigned as one of the node proteins in the protein-protein interaction network and the key protein involved in focal adhesion pathway by bioinformatics analysis. Further biological experiments confirmed that FOXP3 directly bound to the promoter regions of CAV1 to positively regulate CAV1 transcription in GC cells. In summary, our study suggested that FOXP3 can be considered as a tumor suppressor in GC via positively regulating CAV1 through transcriptional activation, and this FOXP3-CAV1 transcriptional regulation axis may play an important role in inhibiting invasion and metastasis of GC cells. Data are available via ProteomeXchange under identifier PXD007725.