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Article ; Online: Investigation of solubilization and digestion methods for microsomal membrane proteome analysis using data-independent LC-MSE.

Mbeunkui, Flaubert / Goshe, Michael B

2011 Volume 11, Issue 5, Page(s) 898–911

Abstract: To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters ... ...

Abstract	To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data-independent LC-MS(E) analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC-MS(E) . These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom-up analysis of membrane proteomes than can be achieved using only a single denaturant.
MeSH term(s)	Chromatography, Liquid ; Guanidine/chemistry ; Lycopersicon esculentum/chemistry ; Membrane Proteins/analysis ; Membrane Proteins/chemistry ; Membrane Proteins/metabolism ; Methanol/chemistry ; Microsomes/chemistry ; Microsomes/enzymology ; Peptide Fragments/analysis ; Peptide Fragments/chemistry ; Peptide Fragments/metabolism ; Plant Roots/chemistry ; Plant Roots/cytology ; Proteome/analysis ; Proteome/chemistry ; Proteome/metabolism ; Proteomics/methods ; Solubility ; Surface-Active Agents/chemistry ; Tandem Mass Spectrometry ; Trypsin/metabolism
Chemical Substances	Membrane Proteins ; Peptide Fragments ; Proteome ; Surface-Active Agents ; Trypsin (EC 3.4.21.4) ; Guanidine (JU58VJ6Y3B) ; Methanol (Y4S76JWI15)
Language	English
Publishing date	2011-03
Publishing country	Germany
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2032093-0
ISSN	1615-9861 ; 1615-9853
ISSN (online)	1615-9861
ISSN	1615-9853
DOI	10.1002/pmic.200900698
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Book ; Online ; Thesis: The effects of low nitrate levels on the freshwater cyanobacterium Synechocystis sp. strain PCC 6803

Mbeunkui, Flaubert

construction of a bioreporter assay and molecular characterization by transcriptome and proteome analysis

2003

Author's details	vorgelegt von Flaubert Mbeunkui
Keywords	Genexpression ; Biosensor ; Nitrate ; Synechocystis ; Wasserblüte ; Proteomanalyse
Language	English
Size	Online-Ressource
Document type	Book ; Online ; Thesis
Thesis / German Habilitation thesis	Univ., Diss--Stuttgart, 2003
Database	Former special subject collection: coastal and deep sea fishing

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Article: Investigation of solubilization and digestion methods for microsomal membrane proteome analysis using data-independent LC-MSE

Mbeunkui, Flaubert / Goshe, Michael B

Proteomics. 2011 Mar., v. 11, no. 5

2011

Abstract	To evaluate the implementation of various denaturants and their efficacy in bottom-up membrane proteomic methods using LC-MS analysis, microsomes isolated from tomato roots were treated with MS-compatible surfactants (RapiGest SF Surfactant from Waters and PPS Silent Surfactant from Protein Discovery), a chaotropic reagent (guanidine hydrochloride), and an organic solvent (methanol). Peptides were analyzed in triplicate sample and technical replicates by data-independent LC-MSE analysis. Overall, 2333 unique peptides matching to 662 unique proteins were detected with the order of denaturant method efficacy being RapiGest SF Surfactant, PPS Silent Surfactant, guanidine hydrochloride, and methanol. Using bioinformatic analysis, 103 proteins were determined to be integral membrane proteins. When normalizing the data as a percentage of the overall number of peptides and proteins identified for each method, the order for integral membrane protein identification efficacy was methanol, guanidine hydrochloride, RapiGest SF Surfactant, and PPS Silent Surfactant. Interestingly, only 8% of the proteins were identified in all four methods with the silent surfactants having the greatest overlap at 17%. GRAVY analysis at the protein and peptide level indicated that methanol and guanidine hydrochloride promoted detection of hydrophobic proteins and peptides, respectively; however, trypsin activity in the presence of each denaturant was determined as a major factor contributing to peptide identification by LC-MSE. These results reveal the complementary nature of each denaturant method, which can be used in an integrated approach to provide a more effective bottom-up analysis of membrane proteomes than can be achieved using only a single denaturant.
Language	English
Dates of publication	2011-03
Size	p. 898-911.
Publishing place	Wiley-VCH Verlag
Document type	Article
ZDB-ID	2032093-0
ISSN	1615-9861 ; 1615-9853
ISSN (online)	1615-9861
ISSN	1615-9853
DOI	10.1002/pmic.200900698
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Isolation and structural elucidation of indole alkaloids from Geissospermum vellosii by mass spectrometry.

Mbeunkui, Flaubert / Grace, Mary H / Lila, Mary Ann

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences

2012 Volume 885-886, Page(s) 83–89

Abstract: Alkaloids from the stem bark of Geissospermum vellosii possess a variety of therapeutic properties including antimalarial activities, activity as a sexual stimulant and inhibition of the proliferation of HIV and herpes viruses. Methods currently used to ... ...

Abstract	Alkaloids from the stem bark of Geissospermum vellosii possess a variety of therapeutic properties including antimalarial activities, activity as a sexual stimulant and inhibition of the proliferation of HIV and herpes viruses. Methods currently used to isolate the active components from G. vellosii are time-consuming, labor intensive, and result in low recovery. In addition, there is a lack of sensitive and accurate analytical methods for the structural characterization and identification of alkaloid components in minor quantities. A combination of high performance counter-current chromatography and ESI tandem mass spectrometry (MS(n)) was established to isolate alkaloids from the stem bark of G. vellosii, and study their electrospray ionization mass spectrometry fragmentation behavior. Five indole alkaloids were successfully isolated and identified by nuclear magnetic resonance and mass spectrometry. The multi-stage tandem mass spectrometric data were used to study their fragmentation pattern and set a model for detailed structure characterization of related indole alkaloids. The presence of the even mass fragment ion suggestive of an odd number of nitrogen at m/z 144 corresponding to C(10)H(9)N was characteristic to indole alkaloids. The results of the experiments demonstrated that the combination of high performance counter current chromatography and ESI-MS(n) is a sensitive, selective and effective approach for rapid isolation and characterization of alkaloids from G. vellosii.
MeSH term(s)	Apocynaceae/chemistry ; Chromatography, High Pressure Liquid/methods ; Countercurrent Distribution/methods ; Indole Alkaloids/chemistry ; Indole Alkaloids/isolation & purification ; Molecular Conformation ; Plant Bark/chemistry ; Spectrometry, Mass, Electrospray Ionization/methods ; Tandem Mass Spectrometry/methods
Chemical Substances	Indole Alkaloids
Language	English
Publishing date	2012-02-15
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1180823-8
ISSN	1873-376X ; 0378-4347 ; 1570-0232 ; 1387-2273
ISSN (online)	1873-376X
ISSN	0378-4347 ; 1570-0232 ; 1387-2273
DOI	10.1016/j.jchromb.2011.12.018
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Article ; Online: Cancer and the tumor microenvironment: a review of an essential relationship.

Mbeunkui, Flaubert / Johann, Donald J

Cancer chemotherapy and pharmacology

2008 Volume 63, Issue 4, Page(s) 571–582

Abstract: Purpose: The role of the microenvironment during the initiation and progression of carcinogenesis is now realized to be of critical importance, both for enhanced understanding of fundamental cancer biology, as well as exploiting this source of ... ...

Abstract	Purpose: The role of the microenvironment during the initiation and progression of carcinogenesis is now realized to be of critical importance, both for enhanced understanding of fundamental cancer biology, as well as exploiting this source of relatively new knowledge for improved molecular diagnostics and therapeutics. Methods: This review focuses on: (1) the approaches of preparing and analyzing secreted proteins, (2) the contribution of tumor microenvironment elements in cancer, and (3) the potential molecular targets for cancer therapy. Results: The microenvironment of a tumor is an integral part of its physiology, structure, and function. It is an essential aspect of the tumor proper, since it supplies a nurturing environment for the malignant process. A fundamental deranged relationship between tumor and stromal cells is essential for tumor cell growth, progression, and development of life threatening metastasis. Improved understanding of this interaction may provide new and valuable clinical targets for cancer management, as well as risk assessment and prevention. Non-malignant cells and secreted proteins from tumor and stromal cells are active participants in cancer progression. Conclusions: Monitoring the change in the tumor microenvironment via molecular and cellular profiles as tumor progresses would be vital for identifying cell or protein targets for cancer prevention and therapy.
MeSH term(s)	Extracellular Matrix/pathology ; Humans ; Neoplasm Proteins/physiology ; Neoplasms/physiopathology ; Stromal Cells/pathology
Chemical Substances	Neoplasm Proteins
Language	English
Publishing date	2008-12-14
Publishing country	Germany
Document type	Journal Article ; Review
ZDB-ID	6820-2
ISSN	1432-0843 ; 0344-5704 ; 0943-9404
ISSN (online)	1432-0843
ISSN	0344-5704 ; 0943-9404
DOI	10.1007/s00280-008-0881-9
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Book ; Online ; Thesis: The effects of low nitrate levels on the freshwater cyanobacterium Synechocystis sp. strain PCC 6803

Mbeunkui, Flaubert [Verfasser]

construction of a bioreporter assay and molecular characterization by transcriptome and proteome analysis

2003

Author's details	vorgelegt von Flaubert Mbeunkui
Keywords	Biowissenschaften, Biologie ; Life Science, Biology
Subject code	sg570
Language	English
Document type	Book ; Online ; Thesis
Database	Digital theses on the web

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Article ; Online: Isolation and characterization of flavonols from blackcurrant by high-performance counter-current chromatography and electrospray ionization tandem mass spectrometry.

Mbeunkui, Flaubert / Grace, Mary H / Yousef, Gad G / Lila, Mary Ann

Journal of separation science

2012 Volume 35, Issue 13, Page(s) 1682–1689

Abstract: Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties ... ...

Abstract	Blackcurrant is considered as a natural high-value food raw material and possesses a variety of therapeutic properties. The health benefits of blackcurrant have generally been credited to its high anthocyanin content; however, the therapeutic properties of other minor flavonoids constituents have not yet been investigated due the difficulties related to their isolation. Multiple steps of high-performance counter-current chromatography in combination with ESI tandem mass spectrometry (MS(n)) were successfully used for the preparative isolation of flavonols from blackcurrant extract, to study their electrospray ionization mass spectrometry fragmentation behavior. Seven flavonols, namely myricetin-3-O-rutinoside (145.5 mg), myricetin-3-O-hexoside (79.7 mg), myricetin-3-O-(6″-malonyl)-glucoside (17.4 mg), kaempferol-3-O-glucoside (20.5 mg), quercetin-3-O-rutinoside (55.1 mg), quercetin-3-O-hexoside (25.8 mg), and myricetin (129.1 mg) have been successfully isolated and their multistage MS(n) data were used for detailed structure characterization. The results of these experiments demonstrated that high-performance counter-current chromatography along with ESI-MS(n) is a sensitive, selective, and effective technology for isolation and characterization of minor constituents from a complex mixture.
MeSH term(s)	Countercurrent Distribution/methods ; Flavonols/chemistry ; Flavonols/isolation & purification ; Plant Extracts/chemistry ; Plant Extracts/isolation & purification ; Ribes/chemistry ; Spectrometry, Mass, Electrospray Ionization/methods
Chemical Substances	Flavonols ; Plant Extracts
Language	English
Publishing date	2012-07
Publishing country	Germany
Document type	Evaluation Studies ; Journal Article
ZDB-ID	2047990-6
ISSN	1615-9314 ; 1615-9306
ISSN (online)	1615-9314
ISSN	1615-9306
DOI	10.1002/jssc.201200198
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Cancer and the tumor microenvironment: a review of an essential relationship

Mbeunkui, Flaubert / Johann, Donald J. Jr

Cancer chemotherapy and pharmacology. 2009 Mar., v. 63, no. 4

2009

Abstract: Purpose The role of the microenvironment during the initiation and progression of carcinogenesis is now realized to be of critical importance, both for enhanced understanding of fundamental cancer biology, as well as exploiting this source of relatively ... ...

Abstract	Purpose The role of the microenvironment during the initiation and progression of carcinogenesis is now realized to be of critical importance, both for enhanced understanding of fundamental cancer biology, as well as exploiting this source of relatively new knowledge for improved molecular diagnostics and therapeutics. Methods This review focuses on: (1) the approaches of preparing and analyzing secreted proteins, (2) the contribution of tumor microenvironment elements in cancer, and (3) the potential molecular targets for cancer therapy. Results The microenvironment of a tumor is an integral part of its physiology, structure, and function. It is an essential aspect of the tumor proper, since it supplies a nurturing environment for the malignant process. A fundamental deranged relationship between tumor and stromal cells is essential for tumor cell growth, progression, and development of life threatening metastasis. Improved understanding of this interaction may provide new and valuable clinical targets for cancer management, as well as risk assessment and prevention. Non-malignant cells and secreted proteins from tumor and stromal cells are active participants in cancer progression. Conclusions Monitoring the change in the tumor microenvironment via molecular and cellular profiles as tumor progresses would be vital for identifying cell or protein targets for cancer prevention and therapy.
Language	English
Dates of publication	2009-03
Size	p. 571-582.
Publisher	Springer-Verlag
Publishing place	Berlin/Heidelberg
Document type	Article
ZDB-ID	6820-2
ISSN	1432-0843 ; 0344-5704 ; 0943-9404
ISSN (online)	1432-0843
ISSN	0344-5704 ; 0943-9404
DOI	10.1007/s00280-008-0881-9
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: In vitro antiplasmodial activity of indole alkaloids from the stem bark of Geissospermum vellosii.

Mbeunkui, Flaubert / Grace, Mary H / Lategan, Carmen / Smith, Peter J / Raskin, Ilya / Lila, Mary Ann

Journal of ethnopharmacology

2012 Volume 139, Issue 2, Page(s) 471–477

Abstract: Ethnopharmacological relevance: The stem bark of Geissospermum vellosii has been traditionally used by the native population of northern South America to treat malaria. Indole alkaloids have been previously isolated from this plant, but the ... ...

Abstract	Ethnopharmacological relevance: The stem bark of Geissospermum vellosii has been traditionally used by the native population of northern South America to treat malaria. Indole alkaloids have been previously isolated from this plant, but the antiplasmodial constituents have not yet been described. As part of our ongoing investigations of new bioactive compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity of isolated fractions and purified alkaloids from Geissospermum vellosii. Materials and methods: Indole alkaloids were isolated and identified from a methanolic crude extract of Geissospermum vellosii bark using a combination of high performance counter current chromatography, mass spectrometry and nuclear magnetic resonance technologies. The methanolic extract, the crude alkaloid fractions and the purified compounds were tested for in vitro antiplasmodial activity against the chloroquine-sensitive strain of Plasmodium falciparum (D10). Results: An indole alkaloid (4) along with four known indole alkaloids, geissolosimine (1), geissospermine (2), geissoschizoline (3), and vellosiminol (5) were isolated and structure elucidated. The antiplasmodial activity (IC(50)) of the methanolic crude extract was 2.22 μg/mL, while for the isolated compounds it ranged from 0.96 μM to 13.96 μM except for (5) which showed a low activity (157 μM). Geissolosimine (1) showed the highest antiplasmodial activity (0.96 μM). Conclusions: This study provides evidence to support the use of Geissospermum vellosii as an antimalarial agent, as used by the native populations. Geissolosimine (1) is a lead molecular structure for possible antimalarial drug development.
MeSH term(s)	Animals ; Antimalarials/chemistry ; Antimalarials/isolation & purification ; Antimalarials/pharmacology ; Antimalarials/toxicity ; Apocynaceae/chemistry ; CHO Cells ; Cell Survival/drug effects ; Chromatography, High Pressure Liquid ; Cricetinae ; Cricetulus ; Dose-Response Relationship, Drug ; Indole Alkaloids/chemistry ; Indole Alkaloids/isolation & purification ; Indole Alkaloids/pharmacology ; Indole Alkaloids/toxicity ; Inhibitory Concentration 50 ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Molecular Structure ; Parasitic Sensitivity Tests ; Plant Bark ; Plants, Medicinal ; Plasmodium falciparum/drug effects
Chemical Substances	Antimalarials ; Indole Alkaloids
Language	English
Publishing date	2012-01-31
Publishing country	Ireland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	134511-4
ISSN	1872-7573 ; 0378-8741
ISSN (online)	1872-7573
ISSN	0378-8741
DOI	10.1016/j.jep.2011.11.036
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Article ; Online: Comparison of health-relevant flavonoids in commonly consumed cranberry products.

Grace, Mary H / Massey, Aaron R / Mbeunkui, Flaubert / Yousef, Gad G / Lila, Mary Ann

Journal of food science

2012 Volume 77, Issue 8, Page(s) H176–83

Abstract: Unlabelled: The human health benefits from consumption of cranberry products have been associated with the fruits' unique flavonoid composition, including a complex profile of anthocyanins and proanthocyanidins. However, when processed by techniques ... ...

Abstract	Unlabelled: The human health benefits from consumption of cranberry products have been associated with the fruits' unique flavonoid composition, including a complex profile of anthocyanins and proanthocyanidins. However, when processed by techniques such as pressing, canning, concentrating, or drying, a number of these natural components may be compromised or inactivated due to physical separation, thermal degradation, or oxidation. Fresh cranberries were compared to freeze-dried berries and individual fruit tissues (skin and peeled fruit). Products examined included cranberry juices (commercial and prepared from concentrate), cranberry sauces (commercial and homemade), and sweetened-dried cranberries (commercial). Freeze-drying resulted in no detectable losses of anthocyanins or proanthocyanidins from cranberry fruits. Anthocyanins were localized in the skin. Proanthocyanins were higher in the skin than in the flesh, with the exception of procyanidin A-2 dimer which was concentrated in the flesh. Anthocyanins were significantly higher in not-from-concentrate juice than in reconstituted juice from concentrate (8.3 mg and 4.2 mg/100 mL, respectively). Similarly, proanthocyanidins were markedly higher in not-from-concentrate juice compared to juice from concentrate (23.0 mg and 8.9 mg/100 mL, respectively). Homemade sauce contained far higher anthocyanins and proanthocyanidins (15.9 and 87.9 mg/100 g, respectively) than canned sauces processed with whole berries (9.6 and 54.4 mg/100 g, respectively) or jelled-type (1.1 and 16 mg/100 g, respectively). Sweetened-dried cranberries were quite low in anthocyanins (7.9 mg/100 g), but they still retained considerable proanthocyanidins (64.2 mg/100 g). Commercially processed products contained significantly lower levels of polyphenols as compared to fresh and home-processed preparations. Anthocyanins were more sensitive to degradation than proanthocyanidins. Practical application: As cranberry juices and other products are increasingly consumed for their recognized health benefits (including prophylaxis against urinary tract infection), it is relevant to consider how various degrees of commercial and home processing can alter innate levels of the biologically active flavonoids (especially anthocyanins and proanthocyanidins) characteristic to the intact fruits.
MeSH term(s)	Anthocyanins/analysis ; Beverages ; Biflavonoids/analysis ; Catechin/analysis ; Chromatography, High Pressure Liquid ; Flavonoids/analysis ; Food Handling/methods ; Freeze Drying ; Fruit/chemistry ; Plant Extracts/analysis ; Proanthocyanidins/analysis ; Vaccinium macrocarpon/chemistry
Chemical Substances	Anthocyanins ; Biflavonoids ; Flavonoids ; Plant Extracts ; Proanthocyanidins ; procyanidin (4852-22-6) ; Catechin (8R1V1STN48) ; 3-hydroxyflavone (ZTG9LSS5QH)
Language	English
Publishing date	2012-08
Publishing country	United States
Document type	Comparative Study ; Journal Article
ZDB-ID	241615-3
ISSN	1750-3841 ; 0022-1147
ISSN (online)	1750-3841
ISSN	0022-1147
DOI	10.1111/j.1750-3841.2012.02788.x
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