bims-oximas Biomed News
on Oxidative stress and mass spectrometry
Issue of 2020‒05‒10
seventeen papers selected by
Alpesh Thakker
University of the Highlands and Islands

  1. J Lipid Res. 2020 May 05. pii: jlr.RA119000606. [Epub ahead of print]
    Pathmasiri KC, Pergande MR, Tobias F, Rebiai R, Rosenhouse-Dantsker A, Bongarzone ER, Cologna SM.
      Niemann-Pick disease, type C1 (NPC1) is a lipid storage disorder in which cholesterol and glycosphingolipids accumulate in late endosomal/lysosomal compartments because of mutations in the NPC1 gene. A hallmark of NPC1 is progressive neurodegeneration of the cerebellum as well as visceral organ damage; however, the mechanisms driving this disease pathology are not fully understood. Phosphoinositides are phospholipids that play distinct roles in signal transduction and vesicle trafficking. Here, we utilized consensus spectra analysis of MS imaging datasets and orthogonal LC-MS analyses to evaluate the spatial distribution of phosphoinositides and quantify them in cerebellar tissue from Npc1-null mice. Our results suggest significant depletion of multiple phosphoinositide species, including phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP), and bisphosphate (PIP2), in the cerebellum of the Npc1-null mice in both whole-tissue lysates and myelin-enriched fractions. Additionally, we observed altered levels of the regulatory enzyme phosphatidylinositol 4-kinase type 2 α (PI4K2A) in Npc1-null mice. In contrast, the levels of related kinases, phosphatases, and transfer proteins were unaltered in the Npc1-null mouse model as observed by Western blot analysis. Our discovery of phosphoinositide lipid biomarkers for NPC1 opens new perspectives on the pathophysiology underlying this fatal neurodegenerative disease.
    Keywords:  Cholesterol; Genetics; Lipidomics; Mass spectrometry; Niemann-Pick disease; lysosomal storage disorder; mass spectrometry imaging; neurodegeneration; phosphoinositide signaling; phospholipid
  2. Expert Rev Proteomics. 2020 May 07.
    Perez de Souza L, Alseekh S, Brotman Y, Fernie AR.
      Introduction: Metabolomics has become a crucial part of systems biology; however data analysis is still often undertaken in a reductionist way focusing on changes in individual metabolites. Whilst such approaches indeed provide relevant insights into the metabolic phenotype of an organism, the intricate nature of metabolic relationships may be better explored when considering the whole system.Areas covered: This review highlights multiple network strategies that can be applied for metabolomics data analysis from different perspectives including: association networks based on quantitative information, mass spectra similarity networks to assist metabolite annotation and biochemical networks for systematic data interpretation. We also highlight some relevant insights into metabolic organization obtained through the exploration of such approaches.Expert opinion: Network based analysis is an established method that allows the identification of non-intuitive metabolic relationships as well as the identification of unknown compounds in mass spectrometry. Additionally, the representation of data from metabolomics within the context of metabolic networks is intuitive and allows for the use of statistical analysis that can better summarize relevant metabolic changes from a systematic perspective.
    Keywords:  Correlation; Metabolomics; Network; Pathway enrichment; Spectra similarity
  3. Int J Mol Sci. 2020 May 04. pii: E3248. [Epub ahead of print]21(9):
    Markovic M, Ben-Shabat S, Aponick A, Zimmermann EM, Dahan A.
      The aim of this work is to analyze relevant endogenous lipid processing pathways, in the context of the impact that lipids have on drug absorption, their therapeutic use, and utilization in drug delivery. Lipids may serve as biomarkers of some diseases, but they can also provide endogenous therapeutic effects for certain pathological conditions. Current uses and possible clinical benefits of various lipids (fatty acids, steroids, triglycerides, and phospholipids) in cancer, infectious, inflammatory, and neurodegenerative diseases are presented. Lipids can also be conjugated to a drug molecule, accomplishing numerous potential benefits, one being the improved treatment effect, due to joined influence of the lipid carrier and the drug moiety. In addition, such conjugates have increased lipophilicity relative to the parent drug. This leads to improved drug pharmacokinetics and bioavailability, the ability to join endogenous lipid pathways and achieve drug targeting to the lymphatics, inflamed tissues in certain autoimmune diseases, or enable overcoming different barriers in the body. Altogether, novel mechanisms of the lipid role in diseases are constantly discovered, and new ways to exploit these mechanisms for the optimal drug design that would advance different drug delivery/therapy aspects are continuously emerging.
    Keywords:  fatty acid; glyceride; lipid; oral drug absorption; phospholipase A2 (PLA2); phospholipid; prodrug; steroid
  4. J Mass Spectrom. 2020 Apr 14. e4525
    Maccelli A, Cesa S, Cairone F, Secci D, Menghini L, Chiavarino B, Fornarini S, Crestoni ME, Locatelli M.
      Many plants of the genus Allium are widely cultivated and consumed for their nutraceutical and health-enhancing bioactive components effective in many metabolic and infectious diseases. In particular, Allium sativum L. (garlic), the most economically important Allium species, is known to present volatile, comparatively polar sulfur-containing compounds responsible for both the typical garlic aroma and antimicrobial property. More recently, the (moderately) polar portion of garlic metabolome, rich of polyphenols and amino acids, is gaining increasing interest as a source of antioxidants and primary nutrients. In this study, we have explored the chemical diversity of eight different hydroalcoholic extracts obtained by microwave-assisted extraction of white and red crop A. sativum and wild Allium triquetrum, Allium roseum, and Allium ampeloprasum, all originating from the Mediterranean Basin. The aim is to appraise their potential dietetic and healing value through an in-depth chemical characterization and contribute to preserve and exploit natural resources. The multimethodological method applied here is based on an untargeted metabolic profiling by means of high-resolution electrospray ionization Fourier-transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry. More than 850 by ESI(+) and 450 by ESI(-) putative metabolites have been annotated covering all main classes of primary and secondary metabolites, including amino acids, alkaloids, organic and fatty acids, nucleotides, vitamins, organosulfur compounds, and flavonoids. The pigment and polyphenol components have been separated and quantified by a targeted chromatographic high-performance liquid chromatography-photodiode array detector (HPLC-PDA) and CIEL*a*b* colorimetric assay, showing characteristic yellow and red components in each extract, related to a different milieu of anthocyanins and flavonoids as assigned by high-resolution mass spectrometry (MS).
    Keywords:  Allium sativum; FT-ICR mass spectrometry; food analysis; metabolomics; wild Allium species
  5. ChemistryOpen. 2020 May;9(5): 568-572
    Gutmann A, Wesenberg LJ, Peez N, Waldvogel SR, Hoffmann T.
      Most of the active pharmaceutical ingredients like Metoprolol are oxidatively metabolized by liver enzymes, such as Cytochrome P450 monooxygenases into oxygenates and therefore hydrophilic products. It is of utmost importance to identify the metabolites and to gain knowledge on their toxic impacts. By using electrochemistry, it is possible to mimic enzymatic transformations and to identify metabolic hot spots. By introducing charged-tags into the intermediate, it is possible to detect and isolate metabolic products. The identification and synthesis of initially oxidized metabolites are important to understand possible toxic activities. The gained knowledge about the metabolism will simplify interpretation and predictions of metabolitic pathways. The oxidized products were analyzed with high performance liquid chromatography-mass spectrometry using electrospray ionization (HPLC-ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. For proof-of-principle, we present a synthesis of one pyridinated main oxidation product of Metoprolol.
    Keywords:  anodic oxidation; charged tags; drug metabolites; electrochemistry; mass spectrometry
  6. Molecules. 2020 May 01. pii: E2120. [Epub ahead of print]25(9):
    Neves B, Duarte S, Domingues P, Pérez-Sala D, Oliveira MM, Domingues MDR, Melo T.
      Nitrated phospholipids have recently been detected in vitro and in vivo and associated with beneficial health effects. They were identified and quantified in biological samples by lipidomics methodologies using liquid chromatography-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) acquired with the linear ion trap mass spectrometer. Only a few studies have used higher-energy collision dissociation (HCD)-MS/MS in high-resolution Orbitraps to characterize nitrated phosphatidylserines and nitrated cardiolipins, highlighting the marked differences in the fragmentation patterns when using CID or HCD fragmentation methods. In this study, we aimed to evaluate the fragmentation of nitrated phosphatidylcholine and nitrated phosphatidylethanolamine species under HCD-MS/MS. We studied the effect of normalized collision energy (NCE) in the fragmentation pattern to identify the best acquisition conditions and reporter ions to detect nitrated phospholipids. The results showed that the intensity of the typical neutral loss of nitrous acid (HNO2) diminishes with increasing NCE, becoming non-detectable for a higher NCE. Thus, the loss of HNO2 could not be the most suitable ion/fragment for the characterization of nitrated phospholipids under HCD. In HCD-MS/MS new fragment ions were identified, corresponding to the nitrated fatty acyl chains, NO2-RCOO-, (NO2-RCOOH-H2O + H)+, and (NO2-RCOOH + H)+, suggested as potential reporter ions to detect nitrated phospholipids when using the HCD-MS/MS lipidomics analysis.
    Keywords:  higher collision-induced dissociation (HCD); lipidomics; nitration; phospholipids; tandem mass spectrometry
  7. Comput Struct Biotechnol J. 2020 ;18 993-999
    Damiani C, Rovida L, Maspero D, Sala I, Rosato L, Di Filippo M, Pescini D, Graudenzi A, Antoniotti M, Mauri G.
      We present MaREA4Galaxy, a user-friendly tool that allows a user to characterize and to graphically compare groups of samples with different transcriptional regulation of metabolism, as estimated from cross-sectional RNA-seq data. The tool is available as plug-in for the widely-used Galaxy platform for comparative genomics and bioinformatics analyses. MaREA4Galaxy combines three modules. The Expression2RAS module, which, for each reaction of a specified set, computes a Reaction Activity Score (RAS) as a function of the expression level of genes encoding for the associated enzyme. The MaREA (Metabolic Reaction Enrichment Analysis) module that allows to highlight significant differences in reaction activities between specified groups of samples. The Clustering module which employs the RAS computed before as a metric for unsupervised clustering of samples into distinct metabolic subgroups; the Clustering tool provides different clustering techniques and implements standard methods to evaluate the goodness of the results.
    Keywords:  Galaxy; Metabolism; RNA-seq; Sample stratification; TCGA
  8. Anal Bioanal Chem. 2020 May 02.
    Maciążek-Jurczyk M, Bessonneau V, Ings J, Bragg L, McMaster M, Servos MR, Bojko B, Pawliszyn J.
      Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.
    Keywords:  Fish steroid hormones; LC–MS/MS; Thin-film coated blades; Thin-film solid-phase microextraction
  9. Adv Drug Deliv Rev. 2020 Apr 28. pii: S0169-409X(20)30029-6. [Epub ahead of print]
    van Kruining D, Luo Q, van Echten-Deckert G, Mielke MM, Bowman A, Ellis S, Oliveira TG, Martinez-Martinez P.
      Lipids play an important role in neurodegeneration, neuroinflammation, and psychiatric disorders and an imbalance in sphingolipid levels is associated to disease. Although early diagnosis and intervention of these disorders would clearly have favorable long-term outcomes, no diagnostic tests currently exist that can accurately identify people at risk. Reliable prognostic biomarkers that are easily accessible would be beneficial to determine therapy and treatment response in clinical trials. Recent advances in lipidomic investigation methods have greatly progressed the knowledge of sphingolipids in neurodegenerative and psychiatric disorders over the past decades although more longitudinal studies are needed to understand its exact role in these disorders to be used as potential tools in the clinic. In this review, we give an overview of the current knowledge of sphingolipids in neurodegenerative and psychiatric disorders and explore recent advances in investigation methods. Finally, the potential of sphingolipid metabolism products and signaling molecules as potential biomarkers for diagnosis, prognostic, or surrogate markers of treatment response is discussed.
    Keywords:  Biomarkers; Lipidomics; Neurodegeneration; Neuroinflammation; Psychiatric diseases; Sphingolipids; Surrogate markers
  10. J Lipid Res. 2020 May 06. pii: jlr.RA120000829. [Epub ahead of print]
    Pike DP, Vogel MJ, McHowat J, Mikuzis PA, Schulte KA, Ford DA.
      Sepsis is defined as the systemic, dysregulated host immune response to an infection that leads to injury to host organ systems, and, often, death. Complex interactions between pathogens and their hosts elicit microcirculatory dysfunction. Neutrophil myeloperoxidase (MPO) is critical for combating pathogens, but MPO-derived hypochlorous acid (HOCl) can react with host molecular species as well. Plasmalogens are targeted by HOCl, leading to the production of 2-chlorofatty acids (2-CLFAs). 2-CLFAs are associated with human sepsis mortality, decrease in vitroendothelial barrier function, and activate human neutrophil extracellular trap formation. Here, we sought to examine 2-CLFAs in an in vivorat sepsis model. Intraperitoneal cecal slurry sepsis with clinically relevant rescue therapies led to ~73% mortality and evidence of microcirculatory dysfunction. Plasma concentrations of 2-CLFAs assessed 8h after sepsis induction were lower in rats that survived sepsis than in non-survivors. 2-CLFA levels were elevated in kidney, liver, spleen, lung, colon and ileum in septic animals. In vivo, exogenous 2-CLFA treatments increased kidney permeability, and in in vitroexperiments 2-CLFA also increased epithelial surface expression of vascular cell adhesion molecule 1 and decreased epithelial barrier function. Collectively, these studies support a role of free 2-CLFAs as biomarkers of sepsis mortality, potentially mediated, in part, by 2-CLFA-elicited endothelial and epithelial barrier dysfunction.
    Keywords:  2-chlorofatty acid (2-CLFA); Fatty acid; Inflammation; Lipids/Oxidation; Neutrophils; Oxidized lipids; Plasmalogens; barrier function; innate immunity; myeloperoxidase; sepsis
  11. JACC Basic Transl Sci. 2020 Apr;5(4): 360-373
    Ståhle M, Silvola JMU, Hellberg S, de Vries M, Quax PHA, Kroon J, Rinne P, de Jong A, Liljenbäck H, Savisto N, Wickman A, Stroes ESG, Ylä-Herttuala S, Saukko P, Abrahamsson T, Pettersson K, Knuuti J, Roivainen A, Saraste A.
      This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of 18F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.
    Keywords:  18F-FDG, 18F-fluorodeoxyglucose; 18F-fluorodeoxyglucose positron emission tomography; ANOVA, analysis of variance; ApoB, apolipoprotein-B; CFR, coronary flow reserve; HAEC, human aortic endothelial cell; ICAM, intracellular adhesion molecule; IL, interleukin; Ig, immunoglobulin; LDLR, low-density lipoprotein receptor; Lp(a), lipoprotein(a); NO, nitric oxide; OxLDL, oxidized low-density lipoprotein cholesterol; OxPLs, oxidized phospholipids; PC, phosphorylcholine; PC-mAb, human PC antibody; VCAM, vascular cell adhesion molecule; atherosclerosis; coronary flow reserve; inflammation; phosphorylcholine
  12. J Chromatogr A. 2020 Apr 28. pii: S0021-9673(20)30385-X. [Epub ahead of print] 461145
    Bogdanova E, Pugajeva I, Reinholds I, Bartkevics V.
    Keywords:  2D-LC; Emerging mycotoxins; Method development; Mycotoxins; Online heart-cutting two-dimensional liquid chromatography
  13. ACS Omega. 2020 Apr 28. 5(16): 9510-9516
    Mayhew AW, Topping DO, Hamilton JF.
      Electrospray ionization (ESI) is widely used as an ionization source for the analysis of complex mixtures by mass spectrometry. However, different compounds ionize more or less effectively in the ESI source, meaning instrument responses can vary by orders of magnitude, often in hard-to-predict ways. This precludes the use of ESI for quantitative analysis where authentic standards are not available. Relative ionization efficiency (RIE) scales have been proposed as a route to predict the response of compounds in ESI. In this work, a scale of RIEs was constructed for 51 carboxylic acids, spanning a wide range of additional functionalities, to produce a model for predicting the RIE of unknown compounds. While using a limited number of compounds, we explore the usefulness of building a predictor using popular supervised regression techniques, encoding the compounds as combinations of different structural features using a range of common "fingerprints". It was found that Bayesian ridge regression gives the best predictive model, encoding compounds using features designed for activity coefficient models. This produced a predictive model with an R 2 score of 0.62 and a root-mean-square error (RMSE) of 0.362. Such scores are comparable to those obtained in previous studies but without the requirement to first measure or predict the physical properties of the compounds, potentially reducing the time required to make predictions.
  14. Ocul Surf. 2020 Apr 30. pii: S1542-0124(20)30075-6. [Epub ahead of print]
    Kim SW, Rho CR, Kim J, Xie Y, Prince RC, Mustafa K, Potma EO, Brown DJ, Jester JV.
      PURPOSE: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC).METHODS: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function.
    RESULTS: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation.
    CONCLUSIONS: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.
    Keywords:  Autophagy; Cell cycle exit; Human meibomian gland epithelial cell; Meibocyte; PPARγ
  15. J Allergy Clin Immunol. 2020 May 01. pii: S0091-6749(20)30627-8. [Epub ahead of print]
    Stevens WW, Staudacher AG, Hulse KE, Carter RG, Winter DR, Kato A, Suh L, Norton JE, Huang JH, Peters AT, Grammer LC, Price CPE, Conley DB, Shintani-Smith S, Tan BK, Welch KC, Kern RC, Schleimer RP.
      BACKGROUND: Aspirin Exacerbated Respiratory Disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance to medications that inhibit cyclooxygenase-1. AERD patients have more severe upper and lower respiratory tract disease compared to aspirin-tolerant CRSwNP patients. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD.OBJECTIVE: To utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD.
    METHODS: Single-cell RNA sequencing (10X Genomics) was utilized to compare the transcriptional profile of NP cells from AERD and CRSwNP patients and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional RT-PCR. Lipid mediators were measured in sinonasal tissue by mass spectrometry. Localization of various proteins within NP was assessed by immunofluorescence.
    RESULTS: The gene encoding for 15-LO, ALOX15, was significantly elevated in NP of AERD compared to CRSwNP (p<0.05) or control (p<0.001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r=0.56, p<0.001) and were associated with asthma. 15-oxo-ETE, a downstream product of 15-LO, was significantly elevated in CRSwNP (27.93pg/mg tissue) and AERD (61.03pg/mg tissue) NP compared to control (7.17pg/mg tissue, p<0.001). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LO+ epithelium in AERD NP.
    CONCLUSIONS: Epithelial and mast cell interactions, leading to the synthesis of 15-oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway, and to the enhanced sinonasal disease severity observed in AERD.
    Keywords:  15-oxo-eicosatetraenoic acid; AERD; AERD, 15-Lipoxygenase; ALOX15; Aspirin Exacerbated Respiratory Disease; CRSwNP; Chronic sinusitis; Hydroxyprostaglandin dehydrogenase; nasal polyps
  16. Anal Chem. 2020 May 05.
    Uppal SS, Mookherjee A, Harkewicz R, Beasley SE, Bush MF, Guttman M.
      Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium exchange (gHDX), MS is also capable of probing the structure of ions. However, gHDX kinetics can depend strongly on many factors, including laboratory temperature, instrumental conditions, and instrument platform selection. These effects can lead to high variability with gHDX measurements, which has hindered the broader adoption of gHDX for structural MS. Here we introduce an approach for standardizing gHDX measurements using co-sampled standards. Quantifying the exchange kinetics for analytes relative to the exchange kinetics of the standards results in greater accuracy and precision than the underlying absolute measurements. The standardization was found to be effective for several types of analytes including small molecules and intact proteins. A subset of analytes showed deviations in their standardized exchange profiles that are attributed to field heating and the concomitant conformational isomerization. Inclusion of helium during the gHDX process for collisional cooling helps mitigate such variations in exchange kinetics related to ion heating. We anticipate that the outcomes of this research will enable the broader use of gHDX in MS-based workflows for molecular identification and isomer differentiation.
  17. Oncol Lett. 2020 Jun;19(6): 4106-4114
    Jia K, Zhao X, Dang X.
      Early biomarkers for pancreatic cancer (PC) detection are required in order to improve patient outcomes. The present study aimed to identify serum biomarkers for PC diagnosis using proteomics, unveil the underlying pathological mechanisms and provide reliable data for the early diagnosis of PC. Isobaric tags for relative and absolute quantification and two-dimensional-liquid chromatography-tandem mass spectometry were used to compare serum samples from patients with PC and healthy individuals. Mascot and Scaffold were used for raw data processing, and Panther for gene ontology (GO) analysis. Igenuity® Pathway Analysis (IPA) was utilized to assess canonical pathways and protein-protein interactions. In total, 76 differentially expressed proteins were identified. The candidate protein DNA repair protein 50 (RAD50) was elevated in patients with PC compared with healthy individuals. In addition, transforming growth factor-β1 (TGF-β1) and apoptotic protease activating factor 1 (APAF-1) were downregulated in PC. GO analysis revealed that the extracellular matrix was increased in PC, as well as receptor function and enzyme regulation; additionally, reduced nucleic acid binding transcription factor activity was observed. IPA analysis demonstrated that the significantly altered canonic pathways were liver X receptor/retinoid X receptor (RXR) activation, the production of nitric oxide and reactive oxygen species in macrophages, the coagulation system, acute phase response signaling and lipopolysaccharide/interleukin-1 mediated inhibition of RXR function. To conclude, RAD50, TGF-β1 and APAF1 are candidate biomarkers for the diagnosis of early PC. The results from the present study could help identify future therapeutic drugs for PC.
    Keywords:  biomarker; isobaric tags for relative and absolute quantification analysis; mass spectrometry; pancreatic cancer; serum