Stable Isotope Dimethyl Labeling Research Articles

Detection of early prognostic biomarkers for metastasis of Ewing's sarcoma in pediatric patients

AimsEwing's Sarcoma is an extremely aggressive tumor in children. The disease is associated with highly metastatic rate, especially at the time of diagnosis, contributing to a lower survival rate and poor prognosis. The study aimed to identify predictive biomarkers for metastatic Ewing's sarcoma through in-depth analysis of the plasma proteome profile of pediatric Ewing's sarcoma patients. Main methodsPlasma samples from Ewing's sarcoma patients and control individuals were profiled using both shotgun and dimethyl-labeled proteomics analysis. Subsequently, Ewing's sarcoma patients were further stratified according to their metastatic state and chemotherapy response. Western blot was used for validation. Univariate and multivariate analyses were performed to determine proteome metastasis predictors. Receiver operating characteristic (ROC) analysis was done to assess the diagnostic significance of the potential plasma Ewing's sarcoma biomarkers. Key findingsOur results revealed a set of proteins significantly associated with the metastatic Ewing's sarcoma disease profile. These proteins include ceruloplasmin and several immunoglobulins. Additionally, our study disclosed significant differentially expressed proteins in pediatric Ewing's sarcoma, including CD5 antigen-like, clusterin, and dermcidin. Stable isotope dimethyl labeling and western blot further confirmed our results, strengthening the impact of such proteins in disease development. Furthermore, an unbiased ROC curve evaluated and confirmed the predictive power of these biomarker candidates. SignificanceThis study presented potential empirical predictive circulating biomarkers for determining the disease status of pediatric Ewing's sarcoma, which is vital for early prediction.

Sijunzi decoction ameliorates gastric precancerous lesions via regulating oxidative phosphorylation based on proteomics and metabolomics

Ethnopharmacological relevanceSijunzi decoction (SJZD), a traditional Chinese medicine formula, is commonly used in clinical practice for the treatment of gastric precancerous lesions (GPL). However, the mechanism of gastric protection is not fully understood. Aims of the studyThe purpose of this study was to systematically evaluate the efficacy of SJZD in blocking the development of GPL and to reveal the underlying mechanism. MethodsFirst, we established a rat model of GPL, which was induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) combined with an irregular diet and 40% ethanol. The efficacy of SJZD was evaluated based on pathological sections and serum biochemical indices. Then, the pharmacodynamic mechanism of SJZD was revealed by quantitative proteomics based on stable isotope dimethyl labeling. At the same time, the pharmacodynamic mechanism was verified by quantitative metabolomics. In addition, the anti-gastritis effect of SJZD was confirmed by a serum pharmacology method in a cell model, and the functional mechanism was further verified. ResultsWe demonstrated that SJZD could block the development of GPL in the animal model. Proteomics and metabolomics revealed that SJZD blocks GPL development by regulating oxidative phosphorylation (OXPHOS). In addition, the serum pharmacology results showed that SJZD-containing serum (SJZD-CS) could inhibit apoptosis in MNNG-induced GES-1 cells. OXPHOS inhibitors could significantly reduce the protective effect of SJZD-CS. ConclusionSJZD effectively ameliorates GPL, and proteomics and metabolomics revealed that its protective effects are closely related to OXPHOS.

Quantitative detection of RAS and KKS peptides in COVID-19 patient serum by stable isotope dimethyl labeling LC-MS.

Angiotensin and kinin metabolic pathways are reported to be altered by many diseases, including COVID-19. Monitoring levels of these peptide metabolites is important for understanding mechanisms of disease processes. In this paper, we report dimethyl labeling of amines in peptides by addition of formaldehyde to samples and deutero-formaldehyde to internal standards to generate nearly identical isotopic standards with 4 m/z units larger per amine group than the corresponding analyte. We apply this approach to rapid, multiplexed, absolute LC-MS/MS quantitation of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) peptides in human blood serum. Limits of detection (LODs) were obtained in the low pg mL-1 range with 3 orders of magnitude dynamic ranges, appropriate for determinations of normal and elevated levels of the target peptides in blood serum and plasma. Accuracy is within ±15% at concentrations above the limit of quantitation, as validated by spike-recovery in serum samples. Applicability was demonstrated by measuring RAS and KKS peptides in serum from COVID-19 patients, but is extendable to any class of peptides or other small molecules bearing reactive -NH2 groups.

Dynamics of Protein Phosphorylation during Arabidopsis Seed Germination.

Seed germination is critical for early plantlet development and is tightly controlled by environmental factors. Nevertheless, the signaling networks underlying germination control remain elusive. In this study, the remodeling of Arabidopsis seed phosphoproteome during imbibition was investigated using stable isotope dimethyl labeling and nanoLC-MS/MS analysis. Freshly harvested seeds were imbibed under dark or constant light to restrict or promote germination, respectively. For each light regime, phosphoproteins were extracted and identified from dry and imbibed (6 h, 16 h, and 24 h) seeds. A large repertoire of 10,244 phosphopeptides from 2546 phosphoproteins, including 110 protein kinases and key regulators of seed germination such as Delay Of Germination 1 (DOG1), was established. Most phosphoproteins were only identified in dry seeds. Early imbibition led to a similar massive downregulation in dormant and non-dormant seeds. After 24 h, 411 phosphoproteins were specifically identified in non-dormant seeds. Gene ontology analyses revealed their involvement in RNA and protein metabolism, transport, and signaling. In addition, 489 phosphopeptides were quantified, and 234 exhibited up or downregulation during imbibition. Interaction networks and motif analyses revealed their association with potential signaling modules involved in germination control. Our study provides evidence of a major role of phosphosignaling in the regulation of Arabidopsis seed germination.

Determination of the soybean allergen Gly m 6 and its stability in food processing using liquid chromatography–tandem mass spectrometry coupled with stable-isotope dimethyl labelling

A cost-effective method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) coupled with stable-isotope dimethyl labelling was used for the determination of Gly m 6. The validation results revealed that the recoveries and precisions obtained from five spiked levels were in the ranges of 88.8–113.0% and 8.3–22.0%, respectively. The content and stability of the major soybean allergen Gly m 6 in various food processing procedures were evaluated by the quantification results of its surrogate signature peptide. The Gly m 6 content in soybean decreased by 42% after natto fermentation, and by 31% and 35% in pasteurised soymilk and sterilised soymilk, respectively, relative to the raw soymilk. Only 19% of Gly m 6 in raw soymilk was retained in the soymilk film. This study extended the feasibility of dimethyl labelling to soy-based food samples and examined the proteolysis of Gly m 6 in natto fermentation and its thermal instability.

Integrated Strategy for Unbiased Profiling of the Histidine Phosphoproteome.

Histidine phosphorylation (pHis), which plays a key role in signal transduction in bacteria and lower eukaryotes, has been shown to be involved in tumorigenesis. Due to its chemical instability, substoichiometric properties, and lack of specific enrichment reagents, there is a lack of approaches for specific and unbiased enrichment of pHis-proteins/peptides. In this study, an integrated strategy was established and evaluated as an unbiased tool for exploring the histidine phosphoproteome. First, taking advantage of the lower charge states of pHis-peptides versus the non-modified naked peptides at weak acid solution (∼pH 2.7), strong cation exchange (SCX) chromatography was used to differentiate modified and non-modified naked peptides. Furthermore, selective enrichment of the pHis-peptide was performed by applying Cu-IDA beads enrichment. Finally, stable isotope dimethyl labeling was introduced to guarantee high-confidence assignment of pHis-peptides. Using this integrated strategy, 563 different pHis-peptides (H = 1) in 385 proteins were identified from HeLa lysates. Motif analysis revealed that pHis prefers hydrophobic amino acids and has the consensus motif-HxxK, which covered the reports from different approaches. Thus, our method may provide an unbiased and effective tool to reveal histidine phosphoproteome and to study the biological process and function of histidine phosphorylation.

Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS

Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeLC-MS/MS is thus an excellent method for identifying fragmentation. However, the lower reproducibility of gel extraction and nano flow LC-MS/MS can produce inaccurate results in comparative analyses of protein quantification among samples. In this study, a novel GeLC-MS/MS method coupled with stable isotope dimethyl labeling was developed. In the method, a mixture of light- and heavy-labeled samples is loaded onto an SDS-PAGE gel, and proteins with different isotopes in one extracted band are quantitatively analyzed by one-shot injection. This procedure enables accurate determination of the abundance ratio of peptides between two samples, even in cases of low peptide abundance, and it is not affected by the reproducibility of the gel extraction or LC-MS procedures. Therefore, our new GeLC-MS/MS method coupled with stable isotope dimethyl labeling provides high accuracy and comprehensive peptide comparisons, enabling the detection of proteolysis events caused by disease or physiological processes.

LC-MS Quantification of Site-Specific Phosphorylation Degree by Stable-Isotope Dimethyl Labeling Coupled with Phosphatase Dephosphorylation.

Protein phosphorylation is a crucial post-translational modification that plays an important role in the regulation of cellular signaling processes. Site-specific quantitation of phosphorylation levels can help decipher the physiological functions of phosphorylation modifications under diverse physiological statuses. However, quantitative analysis of protein phosphorylation degrees is still a challenging task due to its dynamic nature and the lack of an internal standard simultaneously available for the samples differently prepared for various phosphorylation extents. In this study, stable-isotope dimethyl labeling coupled with phosphatase dephosphorylation (DM + deP) was tried to determine the site-specific degrees of phosphorylation in proteins. Firstly, quantitation accuracy of the (DM + deP) approach was confirmed using synthetic peptides of various simulated phosphorylation degrees. Afterwards, it was applied to evaluate the phosphorylation stoichiometry of milk caseins. The phosphorylation degree of Ser130 on α-S1-casein was also validated by absolute quantification with the corresponding synthetic phosphorylated and nonphosphorylated peptides under a selected reaction monitoring (SRM) mode. Moreover, this (DM + deP) method was used to detect the phosphorylation degree change of Ser82 on the Hsp27 protein of HepG2 cells caused by tert-butyl hydroperoxide (t-BHP) treatment. The results showed that the absolute phosphorylation degree obtained from the (DM + deP) approach was comparable with the relative quantitation resulting from stable-isotope dimethyl labeling coupled with TiO2 enrichment. This study suggested that the (DM + deP) approach is promising for absolute quantification of site-specific degrees of phosphorylation in proteins, and it may provide more convincing information than the relative quantification method.

Quantitative determination of osteopontin in bovine, buffalo, yak, sheep and goat milk by Ultra-high performance liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling

Osteopontin (OPN) is a multifunctional protein present in different tissues, body fluids and milk. Different milk has different level of OPN content. To determine the amount of osteopontin in bovine, buffalo, yak, sheep and goat milk, we developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to detect an osteopontin signature peptide. The signature peptides selected by searching Uniprot database for trypsin digested osteopontin. The sample preparation procedure includes trypsin digestion, dimethyl labeling of tryptic peptides, purification and concentration of labeled tryptic peptide with solid phase extraction. The limit of detection and limit of quantification are 0.5 mg L−1 and 2.0 mg L−1, respectively. The method has satisfactory analytical performance with a linearity of R2 ≥ 0.998, recoveries of 103.7–111.0%, and precision of 1.8–6.2%. It is also validated and successfully applied to quantifying osteopontin content in bovine, buffalo, yak, sheep and goat milk.

Identification of Cytosolic Protein Targets of Catechol Estrogens in Breast Cancer Cells Using a Click Chemistry-Based Workflow

Catechol estrogens (CEs) are known to be toxic metabolites and the initiators of the oncogenesis of breast cancers via forming covalent adducts with DNAs. CEs shall also react with proteins, but their cellular protein targets remain unexplored. Here, we reported the identification of protein targets of CEs in the soluble cytosol of estrogen-sensitive breast cancer cells by multiple comparative proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an improved click chemistry-based workflow. Multiple comparative proteomics composed of an experimental pair (probe versus solvent) and two control pairs (solvent versus solvent and probe versus solvent without enrichment) were studied using stable isotope dimethyl labeling. The use of 4-hydroxyethynylestradiol (4OHEE2) probe with an amide-free linker coupled with on-bead digestion and redigestion of the proteins cleaved from the beads was shown to greatly improve the recovery and identification of CE-adducted peptides. A total of 310 protein targets and 40 adduction sites were repeatedly (n ≥ 2) identified with D/H (probe/solvent) ratio >4 versus only one identified with D/H >4 from the two control pairs, suggesting that our workflow imposes only a very low background. Meanwhile, multiple comparative D/H ratios revealed that CEs may downregulate many target proteins involved in the metabolism or detoxification, suggesting a negative correlation between CE-induced adduction and expression of proteins acting on the alleviation of stress-induced cellular damages. The reported method and data will provide opportunities to study the progression of estrogen metabolism-derived diseases and biomarkers.

Quantitative Proteomic Analysis of the Central Amygdala in Neuropathic Pain Model Rats.

Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central sensitization. Data are available via ProteomeXchange with identifier PXD017473.

Differential quantitative proteomics reveals key proteins related to phenotypic changes of breast cancer cells expressing progesterone receptor A

Progesterone receptor isoforms A and B exert different biological effects in breast cancer cells. Alteration of PRA/PRB ratio is often observed during breast cancer progression. High PRA/PRB ratios in breast cancer patients are associated with resistance to chemotherapy and poor prognosis. While it is well accepted that PRA and PRB regulate different sets of genes, how the expression of PRA and PRB alters breast cancer proteomes has not been fully investigated. To directly investigate the effects of PR isoform expression on the breast cancer proteome, both in the presence and absence of progestin, PRA and PRB were independently stably expressed in T47DC42 PR-null breast cancer cells using a doxycycline (Dox)-regulated promoter. Dox induction dose-dependently increased PRA and PRB expression. Dox-induced PRA and PRB showed normal receptor localization and were transcriptionally active. Differential quantitative proteomic analysis by stable isotope dimethyl labeling was performed to quantitatively examine how PR isoforms altered global breast cancer proteomes. Cells expressing PRA in the absence of progestin were enriched in proteins involved in the TCA cycle and enriched in proteins involved in glycolysis in the presence of progestin, whilst cells expressing PRB in the absence and presence progestin were significantly enriched in proteins involved in the cell cycle and cell apoptosis pathways. This proteomic data revealed a link between PR isoform expression and alteration in cell metabolism, cell proliferation, and apoptosis. The enrichment of proteins involved in the glycolytic pathway in breast cancer cells expressing PRA is consistent with stem cell-like properties, previously reported in PRA-rich breast cancer cells. Moreover, compared to liganded PRB, liganded PRA differentially upregulated proteins involved in chromatin remodeling, such as linker histone H1.2. Silencing H1.2 gene expression suppressed PRA-mediated cell proliferation and promoted G2/M and S phase entry of the cell cycle. Additionally, liganded PRA upregulated the expression of cathepsin D (CTSD) protease, whose expression is associated with poor prognosis in breast cancer patients. Together, our data demonstrated that the expression of PRA or PRB dramatically and differentially altered breast cancer cell proteomes. These isoform-specific changes in the breast cancer proteome will help to explain the distinct phenotypic properties of breast cancer cells expressing different levels of PRA and PRB.

Quantitative proteomics reveal the alterations in the spinalcord after myocardial ischemia‑reperfusion injury in rats.

There is now substantial evidence that myocardial ischemia-reperfusion (IR) injury affects the spinal cord and brain, and that interactions may exist between these two systems. In the present study, the spinal cord proteomes were systematically analyzed after myocardial IR injury, in an attempt to identify the proteins involved in the processes. The myocardial IR injury rat model was first established by cross clamping the left anterior descending coronary artery for 30-min ischemia, followed by reperfusion for 2 h, which resulted in a significant histopathological and functional myocardial injury. Then using the stable isotope dimethyl labeling quantitative proteomics strategy, a total of 2,362 shared proteins with a good distribution and correlation were successfully quantified. Among these proteins, 33 were identified which were upregulated and 57 were downregulated in the spinal cord after myocardial IR injury, which were involved in various biological processes, molecular function and cellular components. Based on these proteins, the spinal cord protein interaction network regulated by IR injury, including apop-tosis, microtubule dynamics, stress-activated signaling and cellular metabolism was established. These heart-spinal cord interactions help explain the apparent randomness of cardiac events and provide new insights into future novel therapies to prevent myocardial I/R injury.

Quantitative proteomic and phosphoproteomic studies reveal novel 5-fluorouracil resistant targets in hepatocellular carcinoma

The development of chemoresistance remains the major obstacles to successful chemotherapy of hepatocellular carcinoma. The molecular mechanisms of drug resistance are complex. Identifying the key markers is crucial for development of therapeutic strategies to overcome resistance. In this study, we employed a cell-line model consisting of the 5-fluorouracil resistant Bel/5-Fu cell line and its parental Bel cell line. Using stable isotope dimethyl labeling combined with high-resolution mass spectrometry, in total, 8272 unique proteins and 22,095 phosphorylation sites with high localization confidence were identified. Our data indicated that the GnRH signaling pathway was involved in acquiring drug resistance, which has not been well elucidated. The western blotting results confirmed that the expression levels of PLCβ3 and PLCβ3 pS1105 in Bel/5-Fu cells were increased as compared to Bel cells. Furthermore, the protein levels of SRC and PKCδ, which could phosphorylate PLCβ3 at ser1105, were higher in Bel/5-Fu cells than in Bel cells. The knockdown of SRC, PKCδ and PLCβ3 increased the susceptibility of Bel/5-Fu cells to 5-Fu. Besides, the increased transcription levels of PLCβ3, PKCδ and SRC were significantly associated with decreased overall survival. Together, our deep proteomic and phosphoproteomic data reveal novel therapeutic targets for attenuating 5-Fu resistance in anti-cancer therapy. SignificanceIt was reported that many hepatocellular carcinoma patients are resistance to 5-Fu. Although some studies related to drug resistance have been reported, the underlying mechanisms were not well elucidated. Unlike many single molecular studies, we focused on the global proteome and phosphoproteome analysis of Bel and Bel5-/Fu cell line using stable isotope dimethyl labeling to identify the previously unrecognized signaling pathway for causing 5-Fu resistance. Our results showed that the phosphorylation levels of PLCβ3 pS1105 and the protein levels of PLCβ3, PKCδ and SRC, which are major components of GnRH signaling pathway were higher in Bel/5-Fu cells than in Bel cells. Furthermore, knockdown of PLCβ3, PKCδ and SRC increased the susceptibility of Bel/5-Fu cells to 5-Fu. Overall, this is the first comprehensive proteomic and phosphoproteomic studies on 5-Fu resistant cell line Bel/5-Fu to identify the potential targets of attenuating chemoresistance in hepatocellular carcinoma.

Mass spectrometry combined with affinity probes for the identification of CP4 EPSPS in genetically modified soybeans.

Sample preparation methods used for genetically modified organisms (GMOs) analysis are often time consuming, require extensive manual manipulation, and result in limited amounts of purified protein, which may complicate the detection of low-abundance GM protein. A robust sample pretreatment method prior to mass spectrometry (MS) detection of the transgenic protein (5-enolpyruvylshikimate-3-phosphate synthase [CP4 EPSPS]) present in Roundup Ready soya is investigated. Liquid chromatography-multiple reaction monitoring tandem MS (nano LC-MS/MS-MRM) was used for the detection and quantification of CP4 EPSPS. Gold nanoparticles (AuNPs) and concanavalin A (Con A)-immobilized Sepharose 4B were used as selective probes for the separation of the major storage proteins in soybeans. AuNPs that enable the capture of cysteine-containing proteins were used to reduce the complexity of the crude extract of GM soya. Con A-sepharose was used for the affinity capture of β-conglycinin and other glycoproteins of soya prior to enzymatic digestion. The methods enabled the detection of unique peptides of CP4 EPSPS at a level as low as 0.5% of GM soya in MRM mode. Stable-isotope dimethyl labeling was further applied to the quantification of GM soya. Both probes exhibited high selectivity and efficiency for the affinity capture of storage proteins, leading to the quantitative detection at 0.5% GM soya, which is a level below the current European Union's threshold for food labeling. The square correlation coefficients were greater than 0.99. The approach for sample preparation is very simple without the need for time-consuming protein prefractionation or separation procedures and thus presents a significant improvement over existing methods for the analysis of the GM soya protein.

Feasibility of Utilizing Stable-Isotope Dimethyl Labeling in Liquid Chromatography⁻Tandem Mass Spectrometry-Based Determination for Food Allergens-Case of Kiwifruit.

Stable-isotope dimethyl labeling is a highly reactive and cost-effective derivatization procedure that could be utilized in proteomics analysis. In this study, a liquid chromatography– tandem mass spectrometry in multiple reaction monitoring mode (LC-MS-MRM) platform for the quantification of kiwi allergens was first developed using this strategy. Three signature peptides for target allergens Act d 1, Act d 5, and Act d 11 were determined and were derivatized with normal and deuterated formaldehyde as external calibrants and internal standards, respectively. The results showed that sample preparation with the phenol method provided comprehensive protein populations. Recoveries at four different levels ranging from 72.5–109.3% were achieved for the H-labeled signature peptides of Act d 1 (SPA1-H) and Act d 5 (SPA5-H) with precision ranging from 1.86–9.92%. The limit of quantification (LOQ) was set at 8 pg mL−1 for SPA1-H and at 8 ng mL−1 for SPA5-H. The developed procedure was utilized to analyze seven kinds of hand-made kiwi foods containing 0.0175–0.0515 mg g−1 of Act d 1 and 0.0252–0.0556 mg g−1 of Act d 5. This study extended the applicability of stable-isotope dimethyl labeling to the economical and precise determination of food allergens and peptides.

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre-Fertilization Processes by Quantitative Proteomics.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

Proteomic Analysis of Zn Depletion/Repletion in the Hormone-Secreting Thyroid Follicular Cell Line FRTL-5.

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO4 fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

Early response of C2C12 myotubes to a sub-cytotoxic dose of hemorrhagic metalloproteinase HF3 from Bothrops jararaca venom

Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by viperid snakes. Snake venom metalloproteinases (SVMPs) of the P-III class are main players of the hemorrhagic effect due to their activities in promoting blood vessel disruption. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, shows a minimum hemorrhagic dose of 240 fmol on rabbit skin. The aim of this study was to assess the effects of a sub-cytotoxic dose of HF3 (50 nM) on the proteomic profile of C2C12 differentiated cells (myotubes) in culture, and on the peptidomic profile of the culture supernatant. Quantitative proteomic analysis using stable-isotope dimethyl labeling showed differential abundance of various proteins including enzymes involved in oxidative stress and inflammation responses. Identification of peptides in the supernatant of HF3-treated myotubes revealed proteolysis and pointed out potential new substrates of HF3, including glyceraldehyde-3-phosphate dehydrogenase, and some damage-associated molecular patterns (DAMPs). These experiments demonstrate the subtle effects of HF3 on muscle cells and illustrate for the first time the early proteolytic events triggered by HF3 on myotubes. Moreover, they may contribute to future studies aimed at explaining the inflammation process, hemorrhage and myonecrosis caused by SVMPs. SignificanceOne of the main features of viperid snake envenomation is myotoxicity at the bite site, which, in turn is often associated with edema, blistering and hemorrhage, composing a complex pattern of local tissue damage. In this scenario, besides muscle cells, other types of cells, components of the extracellular matrix and blood vessels may also be affected, resulting in an outcome of deficient muscle regeneration. The main venom components participating in this pathology are metalloproteinases and phospholipases A2. Muscle necrosis induced by metalloproteinases is considered as an indirect effect related to ischemia, due to hemorrhage resulted from damage to the microvasculature. The pathogenesis of local effects induced by Bothrops venoms or isolated toxins has been studied by traditional methodologies. More recently, proteomic and peptidomic approaches have been used to study venom-induced pathogenesis. Here, in order to investigate the role of metalloproteinase activity in local tissue damage, we asked whether the hemorrhagic metalloproteinase HF3, at sub-cytotoxic levels, could alter the proteome of C2C12 myotubes in culture, thereby providing an insight into the mechanisms for the development of myonecrosis. Our results from mass spectrometric analyses showed subtle, early changes in the cells, including differential abundance of some proteins and proteolysis in the culture supernatant. The data illustrate the potential ability of metalloproteinases to trigger early systemic responses progressing from local cells and up to tissues.

Application of stable isotope dimethyl labeling for MRM based absolute antigen quantification of influenza vaccine

Determining the precursor/product ion pair and optimal collision energy are the critical steps for developing a multiple reaction monitoring (MRM) assay using triple quadruple mass spectrometer for protein quantitation. In this study, a platform consisting of stable isotope dimethyl labeling coupled with triple-quadruple mass spectrometer was used to quantify the protein components of the influenza vaccines. Dimethyl labeling of both the peptide N-termini and the ϵ-amino group of lysine residues was achieved by reductive amination using formaldehyde and sodium cyanoborohydrate. Dimethylated peptides are known to exhibit dominant a1 ions under gas phase fragmentation in a mass spectrometer. These a1 ions can be predicted from the peptide N-terminal amino acids, and their signals do not vary significantly across a wide range of collision energies, which facilitates the determination of MRM transition settings for multiple protein targets. The intrinsic a1 ions provide sensitivity for acquiring MRM peaks that is superior to that of the typical b/y ions used for native peptides, and they also provided good linearity (R2 ≥ 0.99) at the detected concentration range for each peptide. These features allow for the simultaneous quantification of hemagglutinin and neuraminidase in vaccines derived from either embryo eggs or cell cultivation. Moreover, the low abundant ovalbumin residue originated from the manufacturing process can also be determined. The results demonstrate that the stable isotope dimethyl labeling coupled with MRM Mass spectrometry screening of a1 ions (i.e., SIDa-MS) can be used as a high-throughput platform for multiple protein quantification of vaccine products.