Proteome Profiling of Rat Brain Cortical Changes during Early Postnatal Brain Development.

Label-free quantitation (LFQ) was applied to proteome profiling of rat brain cortical development during the early postnatal period. Male and female rat brain extracts were prepared using a convenient, detergent-free sample preparation technique at postnatal days (PND) 2, 8, 15, and 22. The PND protein ratios were calculated using Proteome Discoverer, and the PND protein change profiles were constructed separately for male and female animals for key presynaptic, postsynaptic, and adhesion brain proteins. The profiles were compared to the analogous profiles assembled from the published mouse and rat cortex proteomic data, including the fractionated-synaptosome data. The PND protein-change trendlines, Pearson correlation coefficient (PCC), and linear regression analysis of the statistically significant PND protein changes were used in the comparative analysis of the datasets. The analysis identified similarities and differences between the datasets. Importantly, there were significant similarities in the comparison of the rat cortex PND (current work) vs mouse (previously published) PND profiles, although in general, a lower abundance of synaptic proteins in mice than in rats was found. The male and female rat cortex PND profiles were expectedly almost identical (98-99% correlation by PCC), which also substantiated this LFQ nanoflow liquid chromatography-high-resolution mass spectrometry approach.

Evaluation of a 'plug and play' nanoflow liquid chromatography system for MS-based proteomic characterization of clinical FFPE specimens.

INTRODUCTION: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times. AREAS COVERED: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant. EXPERT OPINION: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.

Comparative Proteomic Analysis of Plant-Pathogen Interactions in Resistant and Susceptible Poplar Ecotypes Infected with Botryosphaeria dothidea.

Poplar are important forestry species in China, but the Botryosphaeria dothidea pathogen causes serious economic losses worldwide. To identify candidate B. dothidea resistance proteins and explore the molecular mechanisms involved in poplar-pathogen interactions, proteomic responses of stem samples from resistant and susceptible poplar ecotypes to B. dothidea were investigated using nanoflow liquid chromatography-tandem mass spectrometry with label-free quantitative analysis. We identified 588 proteins, divided into 21 biological process categories including 48 oxidoreductases, 72 hydrolytic enzymes, 80 metabolic enzymes, and 29 proteins of unknown function. Differential proteome analysis revealed large differences between resistant Populus tomentosa Carr and susceptible Populus beijingensis Hsu ecotypes before and after inoculation. Among 102 identified proteins, 22 were highly upregulated in the resistant genotype but downregulated in the susceptible genotype. Proteins induced in P. tomentosa Carr in response to B. dothidea are associated with plant defenses including oxidoreductase activity (catalase, isocitrate dehydrogenase, and superoxide dismutase), phenylpropanoid biosynthesis and phenylalanine metabolism (alcohol dehydrogenase), photosynthesis (ATP synthase subunit alpha, ATP synthase gamma chain, photosystem I P700 chlorophyll a apoprotein A2, photosystem II CP47 chlorophyll apoprotein), carbon fixation (pyruvate kinase, triosephosphate isomerase, malic enzyme, phosphoglycerate kinase, ribulose-1,5-bisphosphate carboxylase, and ribulose bisphosphate carboxylase small chain), and glycolysis/gluconeogenesis (fructose-bisphosphate aldolase). Kyoto Encyclopedia of Genes and Genomes pathway analysis identified 168 proteins related to metabolic pathways, 41 proteins related to the biosynthesis of phenylpropanoids, and 36 proteins related to the biosynthesis of plant hormones, the biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid, and photosynthesis in response to B. dothidea. Our findings provide insight into plant-pathogen interactions in resistant and susceptible poplar ecotypes infected with B. dothidea and could assist the development of novel strategies for fighting poplar canker disease.

Quantitative Proteomic Analysis of the Orbital Frontal Cortex in Rats Following Extended Exposure to Caffeine Reveals Extensive Changes to Protein Expression: Implications for Neurological Disease.

Caffeine is a plant-derived psychostimulant and a common additive found in a wide range of foods and pharmaceuticals. The orbitofrontal cortex (OFC) is rapidly activated by flavours, integrates gustatory and olfactory information, and plays a critical role in decision-making, with dysfunction contributing to psychopathologies and neurodegenerative conditions. This study investigated whether long-term consumption of caffeine causes changes to behavior and protein expression in the OFC. Male adult Sprague-Dawley rats (n = 8 per group) were treated for 26 days with either water or a 0.6 g/L caffeine solution. Locomotor behavior was measured on the first and last day of treatment, then again after 9 days treatment free following exposure to a mild stressor. When tested drug free, caffeine-treated animals were hyperactive compared to controls. Two hours following final behavioral testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free shotgun proteomics found 157 proteins differentially expressed in the caffeine-drinking rats compared to control. Major proteomic effects were seen for cell-to-cell communication, cytoskeletal regulation, and mitochondrial function. Similar changes have been observed in neurological disorders including Alzheimer's disease, Parkinson's disease, and schizophrenia.

Methamphetamine-induced sensitization is associated with alterations to the proteome of the prefrontal cortex: implications for the maintenance of psychotic disorders.

Repeat administration of psychostimulants, such as methamphetamine, produces a progressive increase in locomotor activity (behavioral sensitization) in rodents that is believed to represent the underlying neurochemical changes driving psychoses. Alterations to the prefrontal cortex (PFC) are suggested to mediate the etiology and maintenance of these behavioral changes. As such, the aim of the current study was to investigate changes to protein expression in the PFC in male rats sensitized to methamphetamine using quantitative label-free shotgun proteomics. A methamphetamine challenge resulted in a significant sensitized locomotor response in methamphetamine pretreated animals compared to saline controls. Proteomic analysis revealed 96 proteins that were differentially expressed in the PFC of methamphetamine treated rats, with 20% of these being previously implicated in the neurobiology of schizophrenia in the PFC. We identified multiple biological functions in the PFC that appear to be commonly altered across methamphetamine-induced sensitization and schizophrenia, and these include synaptic regulation, protein phosphatase signaling, mitochondrial function, and alterations to the inhibitory GABAergic network. These changes could inform how alterations to the PFC could underlie the cognitive and behavioral dysfunction commonly seen across psychoses and places such biological changes as potential mediators in the maintenance of psychosis vulnerability.

Development and performance evaluation of an ultralow flow nanoliquid chromatography-tandem mass spectrometry set-up.

LC-MS/MS is the most commonly used technique for the identification and characterization of proteins. The efficiency of the electrospray process is a critical factor in LC-MS/MS. Despite the benefits associated with very low flow rates for the ionization efficiency, most LC-MS/MS platforms are operated at relatively high flow rates. The purpose of this work was to develop a nano LC system operable at a flow rate of 20 nL/min, applicable for routine analysis in proteomics laboratories. Peptide separation was performed with an analytical column packed with 2 µm porous chromatographic beads, a length of 25 cm and an inner diameter (i.d.) of 25 µm. Practical usability, reproducibility, and overall performance of the system were evaluated with a tryptic peptide mixture generated from HeLa cells. Using 100 ng of sample, we identified on average 3721 protein groups based on 25,699 peptides. We demonstrate that the number of peptides identified with this system increases with decreasing flow rates. Probing the sensitivity of the set-up we analyzed only 10 ng of the sample, identifying an average number of 2042 protein groups based on 11 424 peptides. All MS data have been deposited in the ProteomeXchange with identifier PXD000396 (http://proteomecentral.proteomexchange.org/dataset/PXD000396).

Multiple oxidative modifications on hemoglobin are elevated in breast cancer patients as measured by nanoflow liquid chromatography-tandem mass spectrometry.

Breast cancer is strongly connected with elevated oxidative stress. Oxidative modifications of hemoglobin can serve as biomarkers for monitoring oxidative stress status in vivo. The structure of hemoglobin modifications derived from malondialdehyde (MDA) in human blood hemoglobin exists as N-propenal and dihydropyridine (DHP). This study reports the simultaneous quantification of eleven modified peptides in hemoglobin derived from MDA and advanced histidine oxidation in 16 breast cancer patients and 16 healthy women using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry. The results reveal statistically significant increases in the formation of MDA-derived N-propenal and DHP of lysine and advanced oxidation of histidine in hemoglobin of breast cancer patients with the Mann-Whitney U-test p values < 0.0001 and the AUC of ROC between 0.9277 and 1.0. Furthermore, the elevation in modified peptides is significant in patients with early stages of breast cancer. By measuring these oxidative modifications in hemoglobin from a drop of blood, the role of lipid peroxidation and oxidative stress in breast cancer can be assessed using this sensitive assay.

Proteomic analysis of the dorsal and ventral hippocampus of rats maintained on a high fat and refined sugar diet.

The typical Western diet, rich in high saturated fat and refined sugar (HFS), has been shown to increase cognitive decline with aging and Alzheimer's disease, and to affect cognitive functions that are dependent on the hippocampus, including memory processes and reversal learning. To investigate neurophysiological changes underlying these impairments, we employed a proteomic approach to identify differentially expressed proteins in the rat dorsal and ventral hippocampus following maintenance on an HFS diet. Rats maintained on the HFS diet for 8 weeks were impaired on a novel object recognition task that assesses memory and on a Morris Water Maze task assessing reversal learning. Quantitative label-free shotgun proteomic analysis was conducted on biological triplicates for each group. For the dorsal hippocampus, 59 proteins were upregulated and 36 downregulated in the HFS group compared to controls. Pathway ana-lysis revealed changes to proteins involved in molecular transport and cellular and molecular signaling, and changes to signaling pathways including calcium signaling, citrate cycle, and oxidative phosphorylation. For the ventral hippocampus, 25 proteins were upregulated and 27 downregulated in HFS fed rats. Differentially expressed proteins were involved in cell-to-cell signaling and interaction, and cellular and molecular function. Changes to signaling pathways included protein ubiquitination, ubiquinone biosynthesis, oxidative phosphorylation, and mitochondrial dysfunction. This is the first shotgun proteomics study to examine protein changes in the hippocampus following long-term consumption of a HFS diet, identifying changes to a large number of proteins including those involved in synaptic plasticity and energy metabolism. All MS data have been deposited in the ProteomeXchange with identifier PXD000028.

Multiple oxidative and advanced oxidative modifications of hemoglobin in gastric cancer patients measured by nanoflow LC-MS/MS.

Overproduction of the reactive oxygen, nitrogen, and chlorine species by the immune systems during chronic infection and inflammation can cause structural and functional changes of cellular proteins. The high abundance of hemoglobin in blood makes hemoglobin adducts suitable biomarkers for assessing the damage of these reactive species in the body. In this study, a total of 23 types and sites of modification in human hemoglobin were simultaneously analyzed, including monooxygenation of histidine, tyrosine, methionine, and aspartate, conversion of histidine to aspartate and hydroxyaspartate, as well as chlorination and nitration of tyrosine residues. Hemoglobin was isolated from the blood of the study subjects, digested into peptides, and the extents of these modifications were quantified relative to their parent peptides using nanoflow liquid chromatography nanoelectrospray ionization tandem mass spectrometry under selected reaction monitoring (nanoLC - NSI-MS/MS-SRM). The extents of monooxygenation at ß-His-77 and ß-Tyr-130, chlorination at α-Tyr-24 and ß-Tyr-130, and nitrosylation at α-Tyr-24 were elevated in gastric cancer patients. Conversely, conversion of histidine to aspartate at α-His-20, α-His-50, ß-His-2, ß-His-143, and monooxygenation at ß-His-143 were decreased in gastric cancer patients. The areas under the receiver operating characteristics (AUC of ROC) curve of these ten types and sites of hemoglobin modifications were between 0.7644 and 0.9644. The ratio of conversion of histidine to hydroxyaspartate versus conversion of histidine to aspartate was significantly higher in gastric cancer patients at α-His-20, α-His-50, and ß-His-143 (p < 0.05) with AUC of ROC ranging between 0.7689 and 0.9178. To our knowledge, this is the first report of simultaneous measurement of multiple types of oxidative and advanced oxidative hemoglobin modifications in gastric cancer patients. The results revealed elevated levels of oxidative stress-induced protein damage in gastric cancer patients and the potential of using these modifications of hemoglobin as biomarkers for evaluation of oxidative stress in one drop of blood.

Silac-based quantitative proteomic analysis of Lactobacillus reuteri CRL 1101 response to the presence of selenite and selenium nanoparticles.

Stable isotope labeling in cell culture (SILAC) was applied for the first time on a lactic acid bacterium strain (L. reuteri CRL1101) for analyzing differential protein expression associated to selenite(Na2SeO3) and selenium nanoparticles (SeNPs) exposure. 57 and 47 proteins were found de-regulated by >1,5 fold in presence of selenite and SeNPs, respectively. Only 16 out of 104 proteins differentially expressed were commonly altered by selenite and SeNPs. The use of a clustered heat map allows us to visualize relations between the de-regulated proteins and exposure conditions. We identified a number of proteins involved in diverse functions and biological processes such as metabolism of carbohydrates, selenium and lipids; folding, sorting and degradation; environmental information and processing. In presence of both, selenite and SeNPs, proteins related to selenium metabolism such as cystathione beta-lyase and oxidoreductases (thioredoxine reductase and NAD/FAD oxidoreductase) were over expressed. Interestingly, the over expression of thioredoxin reductase could protect the host from oxidizing compounds. An over expression of phage proteins and chaperones with selenite was observed; this result and the fact that a lower cell count was detected when selenite was added could indicate that this latter Se species has a more deleterious effect than the nanoparticles.

Detection and simultaneous quantification of three smoking-related ethylthymidine adducts in human salivary DNA by liquid chromatography tandem mass spectrometry.

Smoking cigarette increases levels of certain ethylated DNA adducts in certain tissues and urine. Cigarette smoking is a major risk factor of various cancers and DNA ethylation is involved in smoking-related carcinogenesis. Among the ethylated DNA adducts, O(2)-ethylthymidine (O(2)-edT) and the promutagenic O(4)-ethylthymidine (O(4)-edT) are poorly repaired and they can accumulate in vivo. Using an accurate, highly sensitive, and quantitative assay based on stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS), O(2)-edT, N(3)-edT (N(3)-ethylthymidine), and O(4)-edT adducts in human salivary DNA were simultaneous detected and quantified. Saliva is easily accessible and available and it can be a potential target in searching for noninvasive biomarkers. Under the highly selected reaction monitoring (H-SRM) mode, salivary samples from 20 smokers and 13 nonsmokers were analyzed. Starting with 50 µg of DNA isolated from about 3.5 mL of saliva, levels of O(2)-edT, N(3)-edT, and O(4)-edT in 20 smokers' salivary DNA samples were 5.3±6.2, 4.5±5.7, 4.2±8.0 in 10(8) normal nucleotides, respectively, while those in 13 nonsmokers were non-detectable. In addition, statistically significant correlations (p<0.0001) were observed between levels of O(2)-edT and N(3)-edT (γ=0.7388), between levels of O(2)-edT and O(4)-edT (γ=0.8839), and between levels of N(3)-edT, and O(4)-edT (γ=0.7835). To the best of our knowledge, this is the first report of detection and quantification of these three ethylthymidine adducts in human salivary DNA, which might be potential biomarkers for exposure to ethylating agents and possibly for cancer risk assessment.

Noninvasive measurement of smoking-associated N(3)-ethyladenine and N(7)-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry.

Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N(3)-ethyladenine (3-EtAde) and N(7)-ethylguanine (7-EtGua). These two ethylpurines can be depurinated spontaneously and be repaired by enzymes and they have been detected in human urine. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human salivary DNA. These ethylpurines were released from DNA by neutral thermal hydrolysis and then enriched by a solid-phase extraction column before nanoLC-NSI/MS/MS analysis. The detection limits (S/N≥3) of 3-EtA and 7-EtG were 15 fg (92 amol) and 10 fg (56 amol), respectively, injected on-column. The lower quantification limits of 3-EtAde and 7-EtGua were both 100 fg, i.e. 620 and 560 amol, respectively, corresponding to 9.4 and 8.6 adducts in 10(9) normal nucleotides, respectively, starting with as little as 20 µg of DNA isolated from an average of 3 mL of saliva. The mean (±SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6±7.0 and 9.7±5.3 in 10(8) normal nucleotides, respectively, while those of 7-EtGua were 14.1±8.2 and 3.8±2.8 in 10(8) normal nucleotides in smokers and nonsmokers, respectively. Levels of 7-EtGua, but not 3-EtAde, were statistically significantly higher in smokers than in nonsmokers (p<0.0001). Furthermore, salivary 7-EtGua levels are significantly correlated with the number of cigarettes smoked per day as well as with the smoking index. This highly specific and sensitive stable isotope dilution nanoLC-NSI/MS/MS assay might be feasible in measuring 7-EtGua in human salivary DNA as a noninvasive biomarker for DNA damage induced by cigarette smoking.

Identification of pancreatic cancer-associated tumor antigen from HSP-enriched tumor lysate-pulsed human dendritic cells.

PURPOSE: Vaccine strategies utilizing dendritic cells (DCs) to elicit anti-tumor immunity are the subject of intense research. Although we have shown that DCs pulsed with heat-treated tumor lysate (HTL) induced more potent anti-tumor immunity than DCs pulsed with conventional tumor lysate (TL), the underlying molecular mechanism is unclear. In order to explore the molecular basis of this approach and to identify potential antigenic peptides from pancreatic cancer, we analyzed and compared the major histocompatibility complex (MHC) ligands derived from TL- and HTL-pulsed dendritic cells by mass spectrophotometry. MATERIALS AND METHODS: Human monocyte-derived dendritic cells were pulsed with TL or HTL prior to maturation induction. To delineate differences of MHC-bound peptide repertoire eluted from DCs pulsed with TL or HTL, nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) was employed. RESULTS: HTL, but not TL, significantly induced DC function, assessed by phenotypic maturation, allostimulation capacity and IFN-γ secretion by stimulated allogeneic T cells. DCs pulsed with TL or HTL displayed pancreas or pancreatic cancer-related peptides in context of MHC class I and II molecules. Some of the identified peptides had not been previously reported as expressed in pancreatic cancer or cancer of other tissue types. CONCLUSION: Our partial lists of MHC-associated peptides revealed the differences between peptide profiles eluted from HTL-and TL-loaded DCs, implying that induced heat shock proteins in HTL chaperone tumor-derived peptides enhanced their delivery to DCs and promoted cross-presentation by DC. These findings may aid in identifying novel tumor antigens or biomarkers and in designing future vaccination strategies.

Simultaneous quantification of ethylpurine adducts in human urine by stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry.

Ethylating agents contained in cigarette smoke can damage DNA producing ethylated DNA adducts, including N(3)-ethyladenine (3-EtAde) and N(7)-ethylguanine (7-EtGua). In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human urine. These urinary adducts were enriched by a polymeric reversed phase solid-phase extraction column before the nanoLC-NSI/MS/MS analysis. The on-column detection limits (S/N≥3) of 3-EtAde and 7-EtGua were 15fg (92amol) and 10fg (56amol), respectively, while the lower quantification limits of 3-EtAde and 7-EtGua were 930 and 840 amol, respectively. Urinary concentrations of 3-EtAde and 7-EtGua in 21 smokers were 68.6±29.4 and 18.7±13.8pg/mL, respectively. In 20 nonsmokers, concentrations of 3-EtAde and 7-EtGua were 3.5±3.8 and 2.4±3.0pg/mL, respectively. The urinary concentrations of 3-EtAde and 7-EtGua were statistically significantly higher in smokers than in nonsmokers (p<0.0001). Moreover, 3-EtAde and 7-EtGua concentrations are significantly correlated with the number of cigarettes smoked per day and with the smoking index. This highly specific and sensitive assay based on stable isotope dilution nanoLC-NSI/MS/MS assay should be clinically valuable in assessing the possibility of measuring urinary ethylpurines as noninvasive biomarkers for smoking-related cancers in humans.

Identification of Pancreatic Cancer-Associated Tumor Antigen from HSP-Enriched Tumor Lysate-Pulsed Human Dendritic Cells

PURPOSE: Vaccine strategies utilizing dendritic cells (DCs) to elicit anti-tumor immunity are the subject of intense research. Although we have shown that DCs pulsed with heat-treated tumor lysate (HTL) induced more potent anti-tumor immunity than DCs pulsed with conventional tumor lysate (TL), the underlying molecular mechanism is unclear. In order to explore the molecular basis of this approach and to identify potential antigenic peptides from pancreatic cancer, we analyzed and compared the major histocompatibility complex (MHC) ligands derived from TL- and HTL-pulsed dendritic cells by mass spectrophotometry. MATERIALS AND METHODS: Human monocyte-derived dendritic cells were pulsed with TL or HTL prior to maturation induction. To delineate differences of MHC-bound peptide repertoire eluted from DCs pulsed with TL or HTL, nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) was employed. RESULTS: HTL, but not TL, significantly induced DC function, assessed by phenotypic maturation, allostimulation capacity and IFN-gamma secretion by stimulated allogeneic T cells. DCs pulsed with TL or HTL displayed pancreas or pancreatic cancer-related peptides in context of MHC class I and II molecules. Some of the identified peptides had not been previously reported as expressed in pancreatic cancer or cancer of other tissue types. CONCLUSION: Our partial lists of MHC-associated peptides revealed the differences between peptide profiles eluted from HTL-and TL-loaded DCs, implying that induced heat shock proteins in HTL chaperone tumor-derived peptides enhanced their delivery to DCs and promoted cross-presentation by DC. These findings may aid in identifying novel tumor antigens or biomarkers and in designing future vaccination strategies.