Plasma-based proteomic profiling identifies the distinct regulation of proteins in hyperplasia and endometrial cancer.

BACKGROUND: Among gynaecological malignancies, endometrial cancer (EC) is the most prevalent type of uterine cancer affecting women. This study explored the proteomic profiles of plasma samples obtained from EC patients, those with hyperplasia (Hy), and a control group (CO). A combination of techniques, such as 2D-DIGE, mass spectrometry, and bioinformatics, including pathway analysis, was used to identify proteins with modified expression levels, biomarkers and their associated metabolic pathways in these groups. METHODS: Thirty-four patients, categorized into three groups-10 with EC, 12 with Hy, and 12 CO-between the ages of 46 and 75 years old were included in the study. Untargeted proteomic analysis was carried out using two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: In all three groups, 114 proteins that were significantly (p ≤ 0.05 and fold change ≥ 1.5) altered were successfully identified using peptide mass fingerprints (PMFs). Compared with those in the control group (CO), the EC samples had 85 differentially expressed proteins (39 upregulated and 46 downregulated), and in the Hy group, 81 proteins were dysregulated (40 upregulated and 41 downregulated) compared to those in the CO group, while 33 proteins exhibited differential regulation (12 upregulated and 21 downregulated) in the EC plasma samples compared to those in the Hy group. Vitamin D binding protein and complement C3 distinguished Hy and EC from CO with the greatest changes in expression. Among the differentially expressed proteins identified, enzymes with catalytic activity represented the largest group (42.9%). In terms of biological processes, most of the proteins were involved in cellular processes (28.8%), followed by metabolic processes (16.7%). STRING analysis for protein interactions revealed that the significantly differentially abundant proteins in the three groups are involved in three main biological processes: signalling of complement and coagulation cascades, regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs), and plasma lipoprotein assembly, remodelling, and clearance. CONCLUSION: The identified plasma protein markers have the potential to serve as biomarkers for differentiating between EC and Hy, as well as for early diagnosis and monitoring of cancer progression.

Methods comparison of two-dimensional gel electrophoresis for host cell protein characterization.

Two-dimensional electrophoresis (2DE) is a gel-based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS-PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two-dimensional difference gel electrophoresis (2D-DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non-traditional method of 2D-DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP-based applications.

Proteomic changes related to actin cytoskeleton function in the skin of vildagliptin-treated mice.

BACKGROUND: Vildagliptin, a dipeptidyl peptidase-4 inhibitor (DPP-4i) is a widely used type 2 diabetes medication that is associated with an up-to 10-fold increased risk for the development of bullous pemphigoid (BP), an autoimmune skin disease. The mechanism by which vildagliptin promotes the development of BP remains unknown. OBJECTIVE: To elucidate effects of vildagliptin treatment on the mouse cutaneous proteome. METHODS: We analyzed the cutaneous proteome of nondiabetic mice treated for 12 weeks with vildagliptin using label-free shotgun mass spectrometry (MS), two-dimensional difference gel electrophoresis (2D-DIGE), immunohistochemistry, immunoblotting, and quantitative real-time polymerase chain reaction. RESULTS: Although vildagliptin treatment did not cause any clinical signs or histological changes in the skin, separate MS and 2D-DIGE analyses revealed altered cutaneous expression of several proteins, many of which were related to actin cytoskeleton remodeling. Altogether 18 proteins were increased and 40 were decreased in the vildagliptin-treated mouse skin. Both methods revealed increased levels of beta-actin and C->U-editing enzyme APOBEC2 in vildagliptin-treated mice. However, elevated levels of a specific moesin variant in vildagliptin-treated animals were only detected with 2D-DIGE. Immunohistochemical staining showed altered cutaneous expression of DPP-4, moesin, and galectin-1. The changed proteins detected by MS and 2D-DIGE were linked to actin cytoskeleton remodeling, transport, cell movement and organelle assembly. CONCLUSION: Vildagliptin treatment alters the cutaneous proteome of nondiabetic mice even without clinical signs in the skin. Cytoskeletal changes in the presence of other triggering factors may provoke a break of immune tolerance and further promote the development of BP.

Liquid semen storage-induced alteration in the protein composition of turkey (Meleagris gallopavo) spermatozoa.

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

Differential proteomic analysis of liver tissues between male and female C57BL / 6J mice by 2D-DIGE / 中国比较医学杂志

Objective To identify the differential proteomic expressions between the liver tissues of male and female mice, and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry ( MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE, 325 protein spots were differentially expressed (|ratio|≥1. 5, P< 0. 05) between the liver tissues of male and female mice, in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females, 14 and 34 proteins were up-or down-regulated in males, respectively. Among them, 6 differentially expressed proteins were validated by Western blot which confirmed the reliability of 2D-DIGE results. GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component, molecular function and biological process. 6 pathways were significantly different between the liver tissues of males and females depending on KEGG analysis. Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.

Differential proteomic analysis of liver tissues between male and female C57BL / 6J mice by 2D-DIGE / 中国比较医学杂志

Objective To identify the differential proteomic expressions between the liver tissues of male and female mice, and investigate the mechanisms underlying gender differences in liver diseases. Methods Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry ( MALDI-TOF-MS) were used to identify the differentially expressed proteins in the liver tissues of male and female C57BL/6J mice. The differentially expressed proteins were validated by Western blot and further analyzed by bioinformatics, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). Results Among the auto-detected 1767 protein spots by 2D-DIGE, 325 protein spots were differentially expressed (|ratio|≥1. 5, P< 0. 05) between the liver tissues of male and female mice, in which 78 spots were randomly selected for MALDI-TOF-MS identification and finally 48 distinct proteins were obtained. Compared with females, 14 and 34 proteins were up-or down-regulated in males, respectively. Among them, 6 differentially expressed proteins were validated by Western blot which confirmed the reliability of 2D-DIGE results. GO analysis showed that the differentially expressed proteins in the liver tissues of male and female mice are associated to various cellular component, molecular function and biological process. 6 pathways were significantly different between the liver tissues of males and females depending on KEGG analysis. Conclusions The proteomic data and related analysis of the liver tissues of C57BL/6J mice offer crucial clues for elucidating the underlying mechanisms of different gender effects on liver diseases.

Differential proteomic analysis of human genetic prion disease patients in frontal lobe tissues / 中华实验和临床病毒学杂志

Objective@#To search for biomarkers for human familial prion disease.@*Methods@#Two-dimensional differential gel electrophoresis (2D-DIGE) proteomic analysis has been performed in frontal lobe tissues of 3 patients suffering from human familial prion disease (PrP) and 3 age-and sex-matched patients suffering from sudden death due to heart failure without neurological disease.@*Results@#The maps revealed 14 polypeptide chains differentially modulated in the PrP samples, among those, 7 could be identified upon digestion and MALDI-TOF/MS analysis, of which 6 appeared to be up-regulated, 1 being down-regulated.@*Conclusions@#We highlight Galectin-1(Gal-1), ryanodine receptor 2 (RyR2), ubiquitin, Rab-interacting lysosomes protein-like protein 1 (RILPL-1) profillin 2 (PFN2), in the differential map. These proteins are related to neurogenesis, the clearance of misfolded proteins, stasis of calium channel, myoclonus and so on. These proteins are potential biomarkers or targets for treatment of prion disease.

Effects of differential expression saliva proteins on OLP patients with Huashi Xingyu Qingre decoction treatment / 实用医学杂志

Objective To analyze the effect of Huashi Xingyu Qingre decoction therapy through identification of the differentially expressed saliva proteins of oral lichen planus. Method The saliva of OLP patients before and after treatment were collected. Total saliva proteins were extracted. The differentially expressed saliva proteins were screened by two-dimensional fluorescence difference gel electrophoresis and identified by liquid chromatography-mass spectrometry. The differentially expressed proteins were analyzed by Western-blot. Results Six differentially expressed proteins were identified as salivary amylase, serum albumin, IgM, carbonic anhydrase VI, zinc-α2- glycoprote and sIgA. The expression level of serum albumin, IgM, carbonic anhydrase VI and zinc-α2-glycoprotein after treatment were lower than that before. However, the expression level of sIgA was higher. The differences were statistically significant. Conclusions Some differentially expressed saliva proteins of OLP before and after Huashi Xingyu Qingre decoction therapy are characterized, and they may play a vital part in the occurrence and development of OLP.

Differentially expressed serum proteins in oral lichen planus patients before and after treated with Huashi Xingyu Qingre Decoction / 医学研究生学报

Objective Oral lichen planus ( OLP) is a common complaint in the oral mucosa , for which there is no definite therapy hitherto.This article aimed to investigate the possible effect of Huashi Xingyu Qingre Decoction (HXQD) on OLP. Methods This study included 30 randomly selected cases of OLP treated with HXQD .Fasting venous blood and total serum protein were obtained before and after medication for screening and identification of OLP-related differential proteins by two-dimensional fluorescence differ-ence gel electrophoresis (2D DIGE) and liquid chromatography-mass spectrometry (LC-MS), followed by Western blot validation . Results Haptoglobin , antithrombin Ⅲ, C1 complement , and vitamin D binding protein were differentially expressed in the serum of the OLP patients before and after treated with HXQD .Compared with the baseline , the expression of haptoglobin was significantly in-creased (103.086 ±27.536 vs 159.704 ±24.228, P<0.05) while that of antithrombin Ⅲ remarkably decreased after treatment (150.00 ±54.04 vs 98.00 ±28.04, P<0.05). Conclusion Haptoglobin, antithrombin Ⅲ, C1 complement, and vitamin D binding protein are differentially expressed in the serum of OLP patients before and after HXQD medication , which may be associated with the development and progression of OLP .

Screening of differentialy expressed saliva proteins from oral lichen planus patients by two-dimensional fluo-rescence difference gel electrophoresis and mass spectrometry / 实用口腔医学杂志

Objective:To identify differentially expressed saliva proteins of oral lichen planus(OLP)patients by two-dimensional fluo-rescence difference electrophoresis(2-D DIGE)and mass spectrometry(MS).Methods:3 pairs of saliva samples from OLP patients and matched healthy adults were collected.Saliva proteins were separated by 2-D DIGE and identified by liquid chromatography-mass spectrometry(LC-MS).Results:SDS-PAGE examination showed that the electrophoresis bands were clear and protein loss was rare. Protein dots were highly reproducible by 2-D DIGE.In average,the abundance of (31 7 ±71 )saliva protein spots were found in OLP pa-tients.4 highly reproducible spots were identified to be secretory IgA1 ,zincα-2-glycoprotein,salivary amylase and serum albumin by LC-MS and they were at higher level in OLP patients than those in the healthy controls.Conclusion:Secretory IgA1 ,zincα-2-glyco-protein,salivary amylase and serum albumin are highly expressed in the saliva of OLP patients,and may be related to the occurrence and development of oral lichen planus.

Proteomic analysis of hepatocellular carcinoma HepG2 cells treated with platycodin D / 中国天然药物

Platycodin D (PD), a triterpenoid saponin isolated from Platycodonis Radix, is a famous Chinese herbal medicine that has been shown to have anti-proliferative effects in several cancer cell lines. The aim of this study was to determine the changes in cellular proteins after the treatment of hepatocellular carcinoma HepG2 cells with PD using proteomics approaches. The cell viability was determined using the MTT assay. The proteome was analyzed by two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot analysis was used to confirm the expression of changed proteins. Our results showed that PD inhibited the proliferation of HepG2 cells in concentration- and time-dependent manners. Sixteen proteins were identified to be up-regulated in PD-treated HepG2 cells, including ATP5H, OXCT1, KRT9, CCDC40, ERP29, RCN1, ZNF175, HNRNPH1, HSP27, PA2G4, PHB, BANF1, TPM3, ECH1, LGALS1, and MYL6. Three proteins (i.e., RPS12, EMG1, and KRT1) decreased in HepG2 cells after treatment with PD. The changes in HSP27 and PHB were further confirmed by Western blotting. In conclusion, our results shed new lights on the mechanisms of action for the anti-cancer activity of PD.

Early diabetic nephropathy plasma proteomic analysis / 重庆医学

Objective To explore the sensitive blood plasma molecular markers in diabetic nephropathy (DN) .Methods Two-di-mensional fluorescence difference gel electrophoresis (2D-DIGE) was used to analyze early DN patients plasma (n=8) and normal plasma ,proteins that showed differential expression of a 1 .5 fold change were analyzed by MALDI-TOF/TOF mass spectrometry . Results 2D-DIGE maps of plasma proteins in patients with DN and normal plasma were established successfully .We validated 13 differentially expressed proteins detected by 2D-DIGE ,including complement component C3、complement component C4、apolipopro-tein E ,etc .Conclusion Proteomic analysis can objectively revealed the differences of protein expression between the two kinds of blood plasma .The 13 proteins might be the potential plasma molecular markers for the clinical diagnosis of diabetic nephropathy .

Quantitative Proteomics Analysis of LCM Purified Stroma of Nasopharyngeal Carcinoma and Normal Nasopharyngeal Mucosa / 生物化学与生物物理进展

The mechanism of how stroma plays an important role in tumor carcinogenesis is now a hotspot. To delineate the features of stromal protein between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal mucosa(NNM), laser capture microdissection (LCM) was performed to purify stromal cells from the NPC and NNM, respectively. The protein expressed profiles of the stroma of NPC and NNM were compared using fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) and 34 differential protein spots between tumor stroma and normal stroma were chosen to be identified by mass spectrometry (MS). A total of 20 differential proteins were identified, and three differential proteins (CapG, L-plastin and S100A9) were selectively further validated by Western blotting and immunohistochemical analysis to confirm the results of 2D-DIGE. 2D-DIGE patterns of the stroma of NPC and NNM were established for the first time, the results suggested that differentially expressed proteins in the stroma of NPC and NNM may be useful for understanding the relationship between NPC cells and their surrounding microenvironment. Further studying of these proteins will be helpful to elucidate the mechanisms of NPC carcinogenesis and provide new thoughts on therapy of NPC through stroma.