bims-prodis 2018-04-15 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2018–04–15
nine papers selected by
Nancy Gough, Bioserendipity

Alendronate and FTI-277 combination as a possible therapeutic approach for hepatocellular carcinoma: An in vitro study.
The depressed central carbon and energy metabolisms is associated to the acquisition of levofloxacin resistance in Vibrio alginolyticus.
pH gradient-liquid chromatography tandem mass spectrometric assay for determination of underivatized polyamines in cancer cells.
Rough Set method accurately predicts unknown protein class/family of Leishmania donovani membrane proteome.
Exosomes secreted by placental stem cells selectively inhibit growth of aggressive prostate cancer cells.
Lipidomic profiling of plasma samples from patients with mitochondrial disease.
The utility of ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) for clinically relevant steroid analysis.
Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics.
A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome.

Hepatobiliary Pancreat Dis Int. 2018 Mar 24. pii: S1499-3872(18)30065-1. [Epub ahead of print]

Alendronate and FTI-277 combination as a possible therapeutic approach for hepatocellular carcinoma: An in vitro study.

Amber Ilyas, Zehra Hashim, Iffat Saeed Channa, Shamshad Zarina.

   BACKGROUND: An important product of mevalonate pathway is downstream synthesis of isoprenoid units that has long been implicated in development and progression of tumor. It has been speculated that inhibition of protein prenylation might be therapeutically beneficial. The objective of current study was to evaluate antitumor potential of a novel therapeutic combination of mevalonate pathway inhibitors, FTI-277 and alendronate. We also examined differentially expressed proteins in response to treatment using proteomics approach.
METHOD: Huh-7 cells were incubated with different concentrations of FTI-277 alone and in combination with alendronate. Differential protein and gene expression was examined through two dimensional gel electrophoresis and real-time quantitative polymerase chain reaction (qPCR), respectively. Proteins were identified using tandem mass spectrometry analysis.
RESULT: Treatment of hepatocellular carcinoma (HCC) cell line with FTI-277 alone showed cell death in a time and dose dependent manner while in combination with alendronate, a synergistic apoptotic effect at 24 h was observed. Proteomic studies on the 20 µmol/L FTI-277 and 5 µmol/L alendronate +20 µmol/L FTI-277 treated cells revealed altered expression of different proteins including peroxiredoxin 2 (Prx2), glutathione S transferase 1 (GSTP1), Rho GTPase activating protein (RhoGAP), triosephosphate isomerase (TPI), and heat shock protein 60 (HSP60). Down-regulated expression of Prx2 and GSTP1 in treated cells was also confirmed by real-time qPCR analysis.
CONCLUSIONS: Combined treatment of FTI-277 and alendronate on Huh-7 HCC cells showed cell death suggesting their anticancer potential. Such treatment approaches are likely to offer new therapeutic strategies.

Keywords:  Chemotherapy; Gene expression; Hepatocellular carcinoma; Proteomics; Therapeutics

DOI:  https://doi.org/10.1016/j.hbpd.2018.03.013
J Proteomics. 2018 Apr 05. pii: S1874-3919(18)30145-3. [Epub ahead of print]

The depressed central carbon and energy metabolisms is associated to the acquisition of levofloxacin resistance in Vibrio alginolyticus.

Zhi-Xue Cheng, Man-Jun Yang, Bo Peng, Xuan-Xian Peng, Xiang-Min Lin, Hui Li.

  The overuse and misuse of antibiotics lead to bacterial antibiotic resistance, challenging human health and intensive cultivation. It is especially required to understand for the mechanism of antibiotic resistance to control antibiotic-resistant pathogens. The present study characterized the differential proteome of levofloxacin-resistant Vibrio alginolyticus with the most advanced iTRAQ quantitative proteomics technology. A total of 160 proteins of differential abundance were identified, where 70 were decreased and 90 were increased. Further analysis demonstrated that crucial metabolic pathways like TCA cycle were significantly down-regulated. qRT-PCR analysis demonstrated the decreased gene expression of glycolysis/gluconeogenesis, the TCA cycle, and fatty acid biosynthesis. Moreover, Na(+)-NQR complex gene expression, membrane potential and the adenylate energy charge ratio were decreased, indicating that the decreased central carbon metabolism is associated to the acquisition of levofloxacin resistance. Therefore, the reduced central carbon and energy metabolisms form a characteristic feature as fitness costs of V. alginolyticus in resistance to levofloxacin.
BIOLOGICAL SIGNIFICANCE: The overuse and misuse of antibiotics lead to bacterial antibiotic resistance, challenging human health and intensive cultivation. Understanding for the antibiotic resistance mechanisms is especially required to control these antibiotic-resistant pathogens. The present study characterized the differential proteome of levofloxacin-resistant Vibrio alginolyticus using the most advanced iTRAQ quantitative proteomics technology. A total of 160 differential abundance of proteins were identified with 70 decreases and 90 increases by liquid chromatography matrix assisted laser desorption ionization mass spectrometry. Most interestingly, crucial metabolic pathways such as the TCA cycle sharply fluctuated. This is the first report that the reduced central carbon and energy metabolisms form a characteristic feature as a mechanism of V. alginolyticus in resistance to levofloxacin.

Keywords:  Antibiotic resistance; Central carbon metabolism; Central energy metabolism; Levofloxacin; Vibrio alginolyticus

DOI:  https://doi.org/10.1016/j.jprot.2018.04.002
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Mar 29. pii: S1570-0232(17)32152-9. [Epub ahead of print]1085 21-29

pH gradient-liquid chromatography tandem mass spectrometric assay for determination of underivatized polyamines in cancer cells.

Hwang Eui Cho, Min H Kang.

  Altered levels of polyamines in biological specimens have been suggested as potential biomarkers for cancer. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase) is reported to modulate polyamines to potentially attenuate proliferation of neuroblastoma cells. A clinical trial is being conducted to evaluate DFMO in various cancers. To determine the pharmacodynamics effect of DFMO, an analytical assay is needed to accurately measure the changes in polyamines in cancer cells. In this study, a novel pH gradient LC-ESI-MS/MS method was developed and validated for the quantitation of polyamines (putrescine, spermidine and spermine) in cancer cells. To separate polar and basic polyamines, a multi-mode column composed of ODS and weak ionic ligands was used. The pH gradient was generated from pH 5.3 to pH 2.7 with 2 mM ammonium acetate and 0.4% acetic acid in 10% acetonitrile as mobile phase. The detection of polyamines was performed utilizing multiple reaction monitoring on electrospray ionization mass spectrometry operated in positive ion mode. A pH gradient method increased resolution and decreased peak width of conventional analytical assays, resulting in 10-250-fold higher detection limits. Mobile phases without ion-pairing reagents were LC-MS compatible and eliminated possible signal suppression and MS contamination. The developed method was successfully applied to the analysis of polyamines in neuroblastoma and leukemia cells treated with DFMO. Putrescine levels were significantly (p < 0.001) decreased in CCRF-CEM (3.68 vs 1.23 ng/mg protein), SK-N-BE(2) (1.98 vs 1.31 ng/mg protein) and CHLA-20 (2.06 vs 0.90 ng/mg protein) cells treated with DFMO relative to vehicle control. The assay will provide a useful tool in determining the pharmacodynamic effect of DFMO in cancer clinical trials.

Keywords:  DFMO; LC-MS/MS; Putrescine; Spermidine; Spermine; pH gradient

DOI:  https://doi.org/10.1016/j.jchromb.2018.03.043
Math Biosci. 2018 Apr 05. pii: S0025-5564(17)30626-0. [Epub ahead of print]

Rough Set method accurately predicts unknown protein class/family of Leishmania donovani membrane proteome.

Arvind Kumar Sinha, Nishant Namdev, Awanish Kumar.

  Leishmania donovani is the primary cause of a fatal disease visceral leishmaniasis (VL) in East Africa and in the Indian subcontinent. Human beings are the only known reservoir of L. donovani and due to the emergence and the spread of drug resistance control for this disease is become worse. Therefore, identification of novel drug target is very important to develop new drug and combat drug resistance issue. Experimental determination of target is costly and time-consuming, hence it is necessary to first identify the efficient target with the accurate mathematical method and then further go for in vitro/in vivo study. Earlier we have predicted the role of protein in term of the target with Naïve Bayes probabilistic classifier on the proteins identified in our L. donovani membrane proteomics study. This time we have used alternative and the popular method named as a Rough Set method (an important part of soft computing method relevance in many real-world applications) and tried to re-visit/validate our earlier findings of L. donovani membrane proteomics and additionally decipher the unknown class/family of membrane proteins as known one. Comparing this result with other classifiers (NB, SVM, RF, C4.5 decision tree) Rough Set method has outperformed and we found the accuracy was 89.28%. This study further validates our previous finding strongly and predicts the class/family of unknown proteins which are very important for the identification and selection toward some novel drug target (still unexplored) and ultimately move in the direction of development of effective antileishmanials.

Keywords:  Drug target; Leishmania donovani; Protein class/family; Rough Set method; Therapy

DOI:  https://doi.org/10.1016/j.mbs.2018.03.027
Biochem Biophys Res Commun. 2018 Apr 09. pii: S0006-291X(18)30801-5. [Epub ahead of print]

Exosomes secreted by placental stem cells selectively inhibit growth of aggressive prostate cancer cells.

Taylor C Peak, Prakash P Praharaj, Gati K Panigrahi, Michael Doyle, Yixin Su, Isabel R Schlaepfer, Ravi Singh, Donald J Vander Griend, Julie Alickson, Ashok Hemal, Anthony Atala, Gagan Deep.

  The current paradigm in the development of new cancer therapies is the ability to target tumor cells while avoiding harm to noncancerous cells. Furthermore, there is a need to develop novel therapeutic options against drug-resistant cancer cells. Herein, we characterized the placental-derived stem cell (PLSC) exosomes (PLSCExo) and evaluated their anti-cancer efficacy in prostate cancer (PCa) cell lines. Nanoparticle tracking analyses revealed the size distribution (average size 131.4 ± 0.9 nm) and concentration of exosomes (5.23 × 1010±1.99 × 109 per ml) secreted by PLSC. PLSCExo treatment strongly inhibited the viability of enzalutamide-sensitive and -resistant PCa cell lines (C4-2B, CWR-R1, and LNCaP cells). Interestingly, PLSCExo treatment had no effect on the viability of a non-neoplastic human prostate cell line (PREC-1). Mass spectrometry (MS) analyses showed that PLSCExo are loaded with 241 proteins and mainly with saturated fatty acids. Further, Ingenuity Pathway Analysis analyses of proteins loaded in PLSCExo suggested the role of retinoic acid receptor/liver x receptor pathways in their biological effects. Together, these results suggest the novel selective anti-cancer effects of PLSCExo against aggressive PCa cells.

Keywords:  Exosomes; Mass spectrometry; Placental stem cells; Prostate cancer; Retinoic acid receptor

DOI:  https://doi.org/10.1016/j.bbrc.2018.04.038
Biochem Biophys Res Commun. 2018 Apr 05. pii: S0006-291X(18)30672-7. [Epub ahead of print]

Lipidomic profiling of plasma samples from patients with mitochondrial disease.

Caixia Ren, Jia Liu, Juntuo Zhou, Hui Liang, Yizhang Zhu, Qingqing Wang, Yinglin Leng, Zhe Zhang, Yun Yuan, Zhaoxia Wang, Yuxin Yin.

  Mitochondrial disease (MD) is a rare mitochondrial respiratory chain disorder with a high mortality and extremely challenging to treat. Although genomic, transcriptomic, and proteomic analyses have been performed to investigate the pathogenesis of MD, the role of metabolomics in MD, particularly of lipidomics remains unclear. This study was undertaken to identify potential lipid biomarkers of MD. An untargeted lipidomic approach was used to compare the plasma lipid metabolites in 20 MD patients and 20 controls through Liquid Chromatography coupled to Mass Spectrometry. Volcano plot analysis was performed to identify the different metabolites. Receiver operating characteristic (ROC) curves were constructed and the area under the ROC curves (AUC) was calculated to determine the potentially sensitive and specific biomarkers. A total of 41 lipids were significantly different in MD patients and controls. ROC curve analysis showed the top 5 AUC values of lipids (phosphatidylinositols 38:6, lysoPC 20:0, 19:0, 18:0, 17:0) are more than 0.99. Multivariate ROC curve based exploratory analysis showed the AUC of combination of top 5 lipids is 1, indicating they may be potentially sensitive and specific biomarkers for MD. We propose combination of these lipid species may be more valuable in predicting the development and progression of MD, and this will have important implications for the diagnosis and treatment of MD.

Keywords:  Biomarkers; Lipidomics; Mitochondrial disease

DOI:  https://doi.org/10.1016/j.bbrc.2018.03.160
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Mar 20. pii: S1570-0232(18)30116-8. [Epub ahead of print]1085 36-41

The utility of ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) for clinically relevant steroid analysis.

Karl-Heinz Storbeck, Lorna Gilligan, Carl Jenkinson, Elizabeth S Baranowski, Jonathan L Quanson, Wiebke Arlt, Angela E Taylor.

  Liquid chromatography tandem mass spectrometry (LC-MS/MS) assays are considered the reference standard for serum steroid hormone analyses, while full urinary steroid profiles are only achievable by gas chromatography (GC-MS). Both LC-MS/MS and GC-MS have well documented strengths and limitations. Recently, commercial ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) systems have been developed. These systems combine the resolution of GC with the high-throughput capabilities of UHPLC. Uptake of this new technology into research and clinical labs has been slow, possibly due to the perceived increase in complexity. Here we therefore present fundamental principles of UHPSFC-MS/MS and the likely applications for this technology in the clinical research setting, while commenting on potential hurdles based on our experience to date.

Keywords:  Gas chromatography–mass spectrometry; Steroid analysis; Ultra-high performance liquid chromatography–tandem mass spectrometry; Ultra-high performance supercritical fluid chromatography–tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jchromb.2018.03.033
J Pharm Biomed Anal. 2018 Apr 02. pii: S0731-7085(18)30405-9. [Epub ahead of print]155 125-134

Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics.

Boglarka Donczo, Andras Guttman.

  More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field.

Keywords:  FFPE; Genomics; Glycomics; Metabolomics; Molecular diagnostics; Proteomics

DOI:  https://doi.org/10.1016/j.jpba.2018.03.065
Redox Biol. 2018 Feb 19. pii: S2213-2317(17)30939-4. [Epub ahead of print]16 359-380

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome.

T R Sutton, M Minnion, F Barbarino, G Koster, B O Fernandez, A F Cumpstey, P Wischmann, M Madhani, M P Frenneaux, A D Postle, M M Cortese-Krott, M Feelisch.

  Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

Keywords:  Glutathione; Hydrogen sulfide; Oxidative stress; Persulfides; Reactive species interactome; Redox status; Thiol-maleimide michael addition

DOI:  https://doi.org/10.1016/j.redox.2018.02.012