iTRAQ Technology Research Articles

Differential proteomes of the cyanobacterium Cyanothece sp. CCY 0110 upon exposure to heavy metals.

The proteomes of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110, grown in medium supplemented with an essential metal (Cu2+) or a non-essential metal (Cd2+),were compared using iTRAQ technology. The data were obtained within a larger study that evaluated the overall effects of different heavy metals on growth/survival, EPS production and ultrastructure of this cyanobacterium [1]. To allow a broader understanding of the strategies triggered to coupe with toxic effects of the metals, Cyanothece′s proteomes were evaluated after chronic and acute exposure to Cu2+ and Cd2+ in two independent 8-plex iTRAQ studies. For the chronic exposure 0.1 mg/l of Cu2+ or 5 mg/l of Cd2+ were used for 10 and 20 days, while in the acute experiments the cells were exposed to 10× these concentrations for 24 h. 202 and 268 proteins were identified and quantified for studies 1 (Cu2+) and 2 (Cd2+), respectively. The majority of the proteins with significant fold changes were associated with photosynthesis, CO2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism.

Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease.

Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology.

Data for transcriptome and proteome analysis of Eucalyptus infected with Calonectria pseudoreteaudii.

Cylindrocladium leaf blight is one of the most important diseases in Eucalyptus plantations. We investigated the proteome and transcriptome of Eucalyptus infected or not infected with Calonectria pseudoreteaudii. Here we provide the information about the processing of raw data obtained by RNA-seq and iTRAQ technologies. The data are related to [1].

Proteomic identification of differentially expressed proteins in rat hepatocarcinogenesis using iTRAQ technology

Proteomic identification of differentially expressed proteins in rat hepatocarcinogenesis using iTRAQ technology

Proteomics Profiles of CD34+ Cells in Bone Marrow of Patients with Severe Aplastic Anemia

ObjectiveApplication of isobaric tags for relative and absolute quantification (iTRAQ) technology, the proteins of CD34+ cells in bone marrow for patients with severe aplastic anemia (SAA) and normal controls were analyzed. To obtain differential proteomics profiles of CD34+ cells in bone marrow for SAA specificity. To preliminary screen the autoantigen which may cause immune system hyperfunction in SAA, providing a theoretical basis for further elucidating the immune pathogenesis of SAA.MethodsUntreated SAA patients, remission SAA patients, as well as normal controls in Tianjin Medical University General Hospital from 2013.1 to 2014.2 were enrolled in this study. CD34+ cells in bone marrow for 29 cases of recovery SAA patients and 10 normal human were purified by immunomagnetic cell sorting, total protein were extracted and were processed enzymolysis, labeled sample by iTRAQ reagent, then analyzed the protein of sample by multidimensional liquid chromatography tandem mass spectrometry of Q Exactive, identified proteins by application of database, compared the different expression of proteins between SAA patients and normal controls, to preliminary screen the autoantigen which may cause immune system hyperfunction in SAA.ResultsExpression of differential proteomic profiles for CD34+ cells in SAA bone marrow were obtained. The protein confidence > 95% and identification of peptide protein false positive rate < 1% were limited, 15120 peptides and 3208 protein were identified. The Ratio of different proteins (SAA group/ normal group) greater than 2.794 and less than 0.323 and the confidence limit greater than 95% were limited , 157 different proteins with high confidence were identified, of which 54 up-regulated proteins, 103 down regulated proteins. The physiological function of proteins involved cell apoptosis, cell cycle, RNA splicing and metabolism, protein modification and transfer, the ubiquitin proteasome mediated protein degradation.ConclusionsThe proteome profiles of CD34+ cells in SAA bone marrow were obtained successfully, and it was proved that iTRAQ marker tandem mass spectrometry for proteome of bone marrow cells for patients with SAA was an effective method. It was found that 157 abnormal expression proteins of CD34+ cells in SAA bone marrow, the function of proteins mainly involved apoptosis, cell cycle, protein modification and transport, RNA processing and splicing, the ubiquitin proteasome system. We preliminary predicted autoantigen which may cause SAA immune "waterfall" activation. DisclosuresNo relevant conflicts of interest to declare.

Proteomic changes during tuber dormancy release process revealed by iTRAQ quantitative proteomics in potato

Given that limited information is available with regard to tuber dormancy release related proteome, we conducted proteome analysis of tuber dormancy release process at dormant tuber (DT), dormancy release tuber (DRT) and sprouting tuber (ST) using the iTRAQ technology. A total of 1,752 proteins were identified. Among them, a subset of 316 proteins was screened as significant up- (137) and down regulated (179) between DT vs DRT. A subset of 120 proteins experienced significant up- (40) or down-regulation (80) between DRT vs ST. The differentially expressed proteins were grouped into 11 functional categories. Proteins enriched in functional categories of major carbohydrate (CHO) metabolism, glycolysis, fermentation, amino acid metabolism, protein and transport were highly up-regulated, while functional categories of photosynthesis and RNA were down-regulated between DT vs DRT. Proteins enriched in functional groups of protein, cell wall, lipid metabolism, miscellaneous, and signaling were strongly up-regulated, while functional categories of photosynthesis, hormone metabolism and protein were down-regulated between DRT vs ST. Consistent with previous documented differentially expressed genes, most of differentially expressed proteins were also identified between DT and DRT, indicating the metabolism shift from growth suspension to growth activation as tubers dormancy breaking. The changes in protein profiles showed lower concordance with corresponding alterations in transcript levels, indicating possible transcriptional and posttranscriptional regulation. Furthermore, the possible mechanism of tuber dormancy release was discussed in relation to what was known in transcripts change and other plant models from carbohydrate metabolism, protein metabolism, stress response, redox regulation, transcription regulation, DNA metabolism, amino acid metabolism, development, signaling as well as hormone metabolism.

ITRAQ-based proteomic analysis of tetramethylpyrazine inhibition on lipopolysaccharide-induced microglial activation

AimsNeurodegenerative diseases are the leading cause of morbidity and mortality worldwide. Several studies have shown that tetramethylpyrazine (TMP) is an effective therapy for neurodegenerative diseases and that it acts by inhibiting the activation of microglial cells in response to inflammatory stimuli. However, the molecular mechanisms underlying the action of TMP remain unknown. Main methodsProteomic analysis was used to generate novel insights into the mechanism by which TMP inhibits microglial activation, and western blotting was used to validate candidate proteins. Key findingsTo identify candidate proteins affected by TMP in lipopolysaccharide-activated microglia, we performed proteomic analysis using iTRAQ labelling coupled with LC TRIPLE-TOF, and we identified 5187 unique proteins. Among these, 266 proteins were differentially expressed and considered putative candidate proteins. Protein annotation revealed that the differentially expressed proteins, such as inducible nitric oxide synthase (iNOS) and ERO1-like protein (ERO1L), might be involved in reducing cellular oxidation in response to stress. Ingenuity pathway analysis revealed that the differentially expressed proteins were involved in a variety of signalling pathways, including liver X receptor/retinoid X receptor (LXR/RXR) activation and the production of nitric oxide and reactive oxygen species in macrophages. Furthermore, one of the differentially expressed protein candidates detected by iTRAQ, iNOS, was confirmed by western blotting. SignificanceOur data suggest that iTRAQ technology is an effective tool to study the mechanism by which TMP inhibits activated microglia. TMP decreased the expression of LXR/RXR-mediated iNOS, which reduced microglial activation in response to inflammatory stimuli.

Online two-dimensional porous graphitic carbon/reversed phase liquid chromatography platform applied to shotgun proteomics and glycoproteomics.

A novel fully automatable two-dimensional liquid chromatography (2DLC) platform has been integrated into a modified commercial off-the-shelf LC instrument, incorporating porous graphitic carbon (PGC) separation and conventional low-pH reversed-phase (RP) separation for both proteomics and N-glycomics analyses; the dual-trap column configuration of this platform offers desirable high-throughput analyses with almost no idle time, in addition to a miniaturized setup and simplified operation. The total run time per analysis was only 19 h when using eight PGC fractions for unattended large-scale qualitative and quantitative proteomic analyses; the identification of 2678 nonredundant proteins and 11,984 unique peptides provided one of the most comprehensive proteome data sets for primary cerebellar granule neurons (CGNs). The effect of pH on the PGC column was investigated for the first time to improve the hydrophobic peptide coverage; the performance of the optimized system was first benchmarked using tryptic digests of Saccharomyces cerevisiae cell lysates and then evaluated through duplicate analyses of Macaca fascicularis cerebral cortex lysates using isobaric tags for relative and absolute quantitation (iTRAQ) technology. An additional plug-and-play PGC module functioned in a complementary manner to recover unretained hydrophilic solutes from the low-pH RP column; synchronization of the fractionations between the PGC-RP system and the PGC module facilitated simultaneous analyses of hydrophobic and hydrophilic compounds from a single sample injection event. This methodology was applied to perform, for the first time, detailed glycomics analyses of Macaca fascicularis plasma, resulting in the identification of a total 130 N-glycosylated plasma proteins, 705 N-glycopeptides, and 254 N-glycosylation sites.

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

Proteomic Analysis of Solid Pseudopapillary Tumor of the Pancreas Reveals Dysfunction of the Endoplasmic Reticulum Protein Processing Pathway

Solid pseudopapillary tumor of the pancreas (SPTP) is a low-grade malignant tumor with a favorable prognosis after surgery. Many previous studies have focused on clinical features or pathological biomarkers of the disease, but a better understanding of the molecular mechanisms underlying SPTP may help guide future therapeutic strategies. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify differentially expressed proteins in SPTP specimens. A total of 1171 proteins with a threshold of a 1.5-fold change and a p value ≤ 0.05 between SPTP tissue and matched normal pancreas tissue were identified for bioinformatics analysis. Mass spectrometry results were then further confirmed by assessing six representative proteins (ACADL, EPHX2, MSI2, DKK4, JUP, and DAD1) in individual specimens with immunohistochemistry. Upon mapping of the differentially expressed proteins to the Kyoto Encyclopedia of Genes and Genomes pathways database, we found several new cell-adhesion molecules that could be used as pathologic biomarkers. Furthermore, we observed that many endoplasmic reticulum-associated proteins were altered, suggesting that endoplasmic reticulum stress may play an important role in SPTP tumorigenesis. Seven proteins (ERO1LB, TRIM1, GRP94, BIP, SEC61B, P4HB, and PDIA4) in this pathway were further validated by immunohistochemistry, and six of them (except SEC61B) coincided to the LC-MS/MS results. This first comprehensive analysis of the SPTP proteome confirms proteins that have been implicated in earlier reports and reveals novel candidates and pathways that could be investigated further for clinical applications.

Abstract 5312: Quantitative proteomic analysis of cell-surface membrane proteins: Biomarker discovery in esophageal squamous cancer

Abstract Background. Esophageal squamous cell cancer (ESCC) is associated with a poor prognosis and high recurrence rate. Identification of proteins that are useful for prognostic assessment and therapeutic targets in ESCC is required. Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels. An analysis of the differential expression of cell-surface membrane proteins would be useful for identification of proteins associated with cell proliferation, cell migration, drug resistance and therapeutic target for antibody-based therapy. Purpose of study. To identify cell-surface membrane proteins overexpressed in ESCC compared with normal esophageal epithelial cells, which may represent novel biomarkers of disease and therapeutic targets. Procedures. We performed a biotinylation-based approach for cell-surface membrane enrichment combined with iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify cell surface proteins overexpressed in ESCC cell lines compared with control normal esophageal epithelial cell line. Results. By combining biotinylation of cell-surface membrane proteins and iTRAQ analysis, 1635 proteins were identified, which contained 402 cell surface membrane proteins. We identified overexpression of 12 membrane proteins, whose expression increased more than twofold in at least four of six ESCC cell lines compared with a normal esophageal epithelial cell line (HEEpic). By this proteomic approach, we identified several tumor antigens which included antigens previously associated with ESCC and antigens not previously reported in this disease. Furthermore, we also successfully identified novel antigen associated with proliferation of ESCC. Cell surface expression of this antigen on ESCC was confirmed by FACS analysis and immunohistochemistry. By transfection of siRNA specific for this novel antigen, significant growth suppression was shown by WST8 assay and induction of apoptosis was detected by caspase-3 assay. Conclusions. By using a high throughput cell-surface membrane proteomic analysis combining biotinylation of cell-surface membrane protein and iTRAQ technology, several ESCC candidate antigens were identified. Further studies on the physiological role of novel tumor antigens overexpressed in ESCC and their application as biomarkers/therapeutic targets are underway. . Note: This abstract was not presented at the meeting. Citation Format: Emi Harada, Satoshi Serada, Tsuyoshi Takahashi, Minoru Fujimoto, Tetsuji Naka. Quantitative proteomic analysis of cell-surface membrane proteins: Biomarker discovery in esophageal squamous cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5312. doi:10.1158/1538-7445.AM2014-5312

Serum differential proteomic analysis of endometriosis and adenomyosis by iTRAQ technique

Objective(s)Adenomyosis and endometriosis are two different diseases, although they have similar pathogenesis and characteristic. The serum differential expressed proteins in adenomyosis and endometriosis may result from the different pathogenesis of two diseases. Proteomic technology is a useful method for detecting all the proteins in samples. We try to use isobaric tags for relative and absolute quantitation (iTRAQ) technology to explore the association between the potential pathogenesis of these two diseases and these identified proteins. Study designThe serum samples from 20 patients with adenomyosis and from 20 patients with endometriosis were analyzed using iTRAQ technology to detect the differential expression of proteins. The validation of the proteins was performed using Western blot. ResultsIn the serum of women with adenomyosis and with endometriosis, 14 proteins were found differentially expressed using iTRAQ technology. Nine proteins were high-expression in adenomyosis group and four proteins increased in endometriosis group. And the differential expression proteins were validated by Western blot. Conclusion(s)The proteins increased in adenomyosis group are related to blood coagulation and complement activation effects, and the proteins high-expression in endometriosis mainly take part in the process of inflammatory response and regulation of apoptosis. The differentially expressed proteins in two groups may due to the different pathogenesis of two diseases.

A quantitative proteomic approach to identify significantly altered protein networks in the serum of patients with lymphangioleiomyomatosis (LAM).

Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition affecting approximately 3.4–7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05). Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, p = 0.03), von Willebrand Factor (40% reduction in LAM, p = 0.03) and Kallikrein III (25% increase in LAM, p = 0.03). Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future.

Comparative proteomic analysis of renal tissue in IgA nephropathy with iTRAQ quantitative proteomics.

Immunoglobulin (Ig) A nephropathy (IgAN) is the most common form of glomerulonephritis. In clinical practice, it is difficult to monitor the repeating relapse in patients suffering from IgAN, which usually occurs within 10 years of end-stage renal disease. In order to identify and quantify the total protein content in the renal tissue of patients with IgAN, isobaric tags for relative and absolute quantification (iTRAQ) technology was performed. iTRAQ coupled with multiple chromatographic fractionation and tandem mass spectrometry was used to analyze the total protein of normal renal tissue in IgAN and healthy patients. The individual proteins were identified by the Mascot search engine and any that were differentially expressed were monitored. A total of 574 different proteins were identified, and 287 proteins were up- or downregulated by >1 fold alteration in levels. The results showed that iTRAQ-based quantitative proteomic technology for the identification and relative quantitation of the renal tissue proteome is efficiently applicable. The differential expression of the proteome profiles for IgAN patients was determined. Further studies using large cohorts of patient samples with long-term clinical follow-up data should be conducted to evaluate the usefulness of the pathogenesis and novel biomarker candidates of IgAN, which may develop a novel technique for the diagnosis of IgAN.

Mitochondrial protein targets of radiosensitisation by 1,8-dihydroxy-3-acetyl-6-methyl-9,10 anthraquinone on nasopharyngeal carcinoma cells

In our preliminary study, 1,8-dihydroxy-3-acetyl-6-methyl-9,10 anthraquinone (GXHSWAQ-1), synthesised according to the basic structure of emodin, exhibited a 1.58-fold radiosensitisation on nasopharyngeal carcinoma CNE-1 cells. This study demonstrated that its radiosensitisation activity was achieved by altering the mitochondrial structure: swollen volume, fragmented crista, and decreasing transmembrane potential (P<0.01). Using isobaric tag for relative and absolute quantitation (iTRAQ) technology, 1396 proteins were identified, and the differentially expressed proteins were involved in metabolism, cell proliferation, angiogenesis, DNA repair process according to the biological process clustering results. Bioinformatic analysis showed that CDH1, RAC1, CDC42 proteins might be mostly mitochondrial targets in the radiosensitisation process. Western blotting analyses verified the differential expression of these proteins.

Identification and quantitative analysis of hepatic protein and its relationship to prognosis in biliary atresia patients by quantitative proteomic technology

Objective To identify and quantify total proteins in liver tissue of patients with biliary atresia (BA) by isobaric tags for relative and absolute quantification (iTRAQ) technology to decipher the relationship between significant protein and its prognosis.Methods According to the serum level of total bilirubin (TBIL) in 3 months and native liver survival time (NLST),the patients were divided into 2 groups of good prognosis (3M TBIL≤ 25 μmol/L,NLST＞2 years) and poor prognosis (3M TBIL＞25μmol/L,NLST＜ 1 year) (n =10 each).Liver tissues were collected during Kasai operation.And iTRAQ technique was used to identify and quantify the expression of differential proteins.Then differential proteins were classified by Panther classification system.The results were further validated by Western blot.Results A total of 5177 proteins were identified by mass spectrometry.Among them,323 differential proteins with expression folds of over 1.5 were identified.Among these proteins,as compared with poor prognosis group,131 proteins were up-regulated and 192 proteins down-regulated in good prognosis group.These differential proteins were related with 14 kinds of biological processes (e.g.metabolic and immune system).Conclusions The technique of iTRAQ is effective for comparative proteomic studies.Thus these identified differential proteins will provide further clues for studying the pathogenesis and prognosis of BA. Key words: Biliary atresia; Mifferential proteins; Prognosis

Cerebrospinal Fluid Biomarker Candidates Associated with Human WNV Neuroinvasive Disease

During the last decade, the epidemiology of WNV in humans has changed in the southern regions of Europe, with high incidence of West Nile fever (WNF) cases, but also of West Nile neuroinvasive disease (WNND). The lack of human vaccine or specific treatment against WNV infection imparts a pressing need to characterize indicators associated with neurological involvement. By its intimacy with central nervous system (CNS) structures, modifications in the cerebrospinal fluid (CSF) composition could accurately reflect CNS pathological process. Until now, few studies investigated the association between imbalance of CSF elements and severity of WNV infection. The aim of the present study was to apply the iTRAQ technology in order to identify the CSF proteins whose abundances are modified in patients with WNND. Forty-seven proteins were found modified in the CSF of WNND patients as compared to control groups, and most of them are reported for the first time in the context of WNND. On the basis of their known biological functions, several of these proteins were associated with inflammatory response. Among them, Defensin-1 alpha (DEFA1), a protein reported with anti-viral effects, presented the highest increasing fold-change (FC>12). The augmentation of DEFA1 abundance in patients with WNND was confirmed at the CSF, but also in serum, compared to the control individual groups. Furthermore, the DEFA1 serum level was significantly elevated in WNND patients compared to subjects diagnosed for WNF. The present study provided the first insight into the potential CSF biomarkers associated with WNV neuroinvasion. Further investigation in larger cohorts with kinetic sampling could determine the usefulness of measuring DEFA1 as diagnostic or prognostic biomarker of detrimental WNND evolution.

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

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.

Proteomic analysis of bladder cancer by iTRAQ after Bifidobacterium infantis-mediated HSV-TK/GCV suicide gene treatment

In our previous studies, we constructed the Bifidobacterium infantis thymidine kinase/nucleoside analogue ganciclovir (BI-TK/GCV) system, which was proven to have a sustainable antitumor activity in an in vivo bladder cancer rodent model. In this article, a proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ) and followed by liquid chromatography-tandem mass spectrometry was used to understand the molecular mechanisms of this system. iTRAQ identified 192 downregulated and 210 upregulated proteins after treatment with BI-TK/GCV in Sprague-Dawley rats. Downregulations of proliferating cell nuclear antigen (PCNA), pyruvate kinase isozymes M2 (PKM2), hexokinase 1 (HXK-1), 6-phosphofructokinase (PFK-B), and cell surface glycoprotein (CD146) in bladder cancer after treatment were confirmed by Western blot analysis and validated by immunohistochemistry. Furthermore, the networks of cancer proliferation associated with PCNA, glycolysis associated with PKM2, HXK-1, and PFK-B, and invasion associated with CD146 were illustrated using Ingenuity Pathway Analysis. This study represents the successful application of iTRAQ technology to reveal the molecular mechanisms of BI-TK/GCV treatment system and provides the theoretical support for the effectiveness of our successful treatment system.

New findings of kinase switching in gastrointestinal stromal tumor under imatinib using phosphoproteomic analysis

Despite the revolutionary effects of imatinib on advanced gastrointestinal stromal tumors (GISTs), most patients eventually develop disease progression following primary resistance or acquired resistance driven by secondary-resistant mutations. Even in radiographically vanishing lesions, pathology has revealed persistent viable cells during imatinib therapy, which could lead to the emergence of drug-resistant clones. To uncover the mechanisms underlying these clinical issues, here we examined imatinib-induced phosphoproteomic alterations in GIST-T1 cells, using our quantitative tyrosine phosphoproteomic analysis method, which combined immunoaffinity enrichment of phosphotyrosine-containing peptides with isobaric tags for relative and absolute quantitation (iTRAQ) technology. Using this approach, we identified 171 tyrosine phosphorylation sites spanning 134 proteins, with 11 proteins exhibiting greater than 1.5-fold increases in tyrosine phosphorylation. Among them, we evaluated FYN and focal adhesion kinase (FAK), both of which are reportedly involved in proliferation and malignant alteration of tumors. We confirmed increased tyrosine phosphorylation of both kinases by western blotting. Inhibition of FYN and FAK phosphorylation each increased tumor cell sensitivity to imatinib. Furthermore, a FAK-selective inhibitor (TAG372) induced apoptosis of imatinib-resistant GIST-T1 cells and decreased the imatinib IC50 . These results indicate that FYN or FAK might be potential therapeutic targets to overcome resistance to imatinib in GISTs. Additionally, we showed that the iTRAQ-based quantitative phosphotyrosine-focused phosphoproteomic approach is a powerful method for screening phosphoproteins associated with drug resistance.