Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods ... more Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods used in their study have utilized low-pressure phenylboronate affinity chromatography materials for the purification of the cisdiol-containing nucleosides. In this study, a boronate HPLC column was surprisingly shown not to trap the nucleosides as would be expected from experience with the classic Affigel 601 resin but showed only partial selectivity toward cis-diol groups while other groups exhibited better retention. In aprotic conditions, trapping of nucleosides was possible; however, the selectivity toward cis-diol-containing compounds was lost with the Lewis basicity of available nitrogens being the main determinant of retention. The experimental findings are compared to and confirmed by DFT calculations.
ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts fo... more ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts formed between 2′—deoxyadenosine, thymidine, 2′—deoxycytidine and 2′—deoxyguanosine with phenylglycidyl ether is described.
ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts fo... more ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts formed between 2′—deoxyadenosine, thymidine, 2′—deoxycytidine and 2′—deoxyguanosine with phenylglycidyl ether is described.
Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-M... more Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.
While protein interaction studies and protein network modeling come to the forefront, the isolati... more While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well-studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co-IP. Furthermore practical issues concerning the use of this 2-D BN/SDS-PAGE display method for the analysis of protein-protein interactions are discussed.
Journal of the American Society for Mass Spectrometry, 2006
In-source collision induced dissociation was applied to access second generation ions of protonat... more In-source collision induced dissociation was applied to access second generation ions of protonated guanosine. The in-source gas-phase behavior of [BH 2 ] ϩ -NH 3 (m/z 135, C 5 H 3 N 4 O ϩ ) was investigated. Adduct formation and reactions with available solvent molecules (H 2 O and CH 3 OH) were demonstrated. Several addition/elimination sequences were observed for this particular ion and solvent molecules. Dissociation pathways for the newly formed ions were developed using a QqTOF mass spectrometer, permitting the assignment of elemental compositions of all product ions produced. Reaction schemes were suggested arising from the ring-opened intermediate of the protonated base moiety [BH 2 ] ϩ , obtained from fragmentation of guanosine. The mass spectral data revealed that the in-source CH 3 OH-reaction product underwent more complex fragmentations than the comparable ion following reaction with H 2 O. A rearrangement and a parallel radical dissociation pathway were discerned. Apart from the mass spectrometric evidence, the fragmentation schemes are supported by density functional theory calculations, in which the reaction of the ring-opened protonated guanine intermediate with CH 3 OH and a number of subsequent fragmentations were elaborated. Additionally, an in-source transition from the ring-opened intermediate of protonated guanine to the ring-opened intermediate of protonated xanthine was suggested. For comparison, a low-energy collision induced dissociation study of xanthosine was performed. Its dissociation pathways agreed with our assumption
Journal of the American Society for Mass Spectrometry, 2006
no. 11 was inadvertently dropped from the Reference section. The publisher apologizes for this er... more no. 11 was inadvertently dropped from the Reference section. The publisher apologizes for this error. The entry for [11] is for Gaussian 03, and should have been provided as below: 11. Gaussian 03, Revision C.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; and Pople, J. A.; Gaussian, Inc., Wallingford CT, 2004.
Journal of the American Society for Mass Spectrometry, 2005
An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source c... more An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source collision-induced dissociation high-mass accuracy tandem mass spectrometry experiment. The equivalent of MS 4 data, a level of information normally achieved on ion trap instruments, was obtained on a Q-TOF mass spectrometer. The combination of the features of high-resolution, accuracy, and in-source CID permitted the unambiguous elucidation of the different fragmentation pathways. Furthermore the elemental compositions of the product ions generated were assigned and their mutual genealogical relationships established. Formerly proposed dissociation pathways of guanosine were revisited and elaborated on more deeply. Furthermore, the presence of H 2 O in the collision cell of several tandem MS instruments was demonstrated and its effect on the product ion spectra investigated. The neutral gain of H 2 O by particular fragments of guanosine was experimentally proven by using argon, saturated with H 2 18 O, as the collision gas. Data indicating the occurrence of more complex reactions in the collision cell as a result of the presence of H 2 O were produced, specifically relating to neutral gain/neutral loss sequences. In silico calculations supported the experimental observation of neutral gain by guanosine fragments and predicted a similar behavior for adenosine. The latter was subsequently experimentally confirmed. (J Am Soc Mass Spectrom 2005, 16, 1291-1304 N ucleosides have been established as molecules of exceptional importance in biochemical research for a long period. They are the building blocks of the macromolecules RNA and DNA, exogeneous nucleoside analogs are used as anti(retro)viral and anticancer agents [1], and nucleoside levels in urine have the potential to act as cancer biomarkers . As their occurrence is both ubiquitous and quantitatively varying [6, 7] their unequivocal characterization is crucial.
A study of the interaction of phosphorylated organic compounds with the stainless components of a... more A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.
Abstract 2′-Deoxycitidine (dCyd) and 2′-deoxyguanosine (dGuo) were subjected to reaction with phe... more Abstract 2′-Deoxycitidine (dCyd) and 2′-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2′-deoxynucleoside adducts. Separation methods were developed on ...
Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum(I1) complex in order to ... more Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum(I1) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5'-mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5'-mononucleotides.
Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 gli... more Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J. Neurochem. 89, 442.]. In the present study, the mechanisms involved in this response were further elucidated. In cells transfected with the Gβγ-scavenger β-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gβγ-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCζ decreased basal and 2MeSADP-stimulated phosphorylation of PKB indicating a role for this PKC isoform in PKB signalling. Although the increased PKB phosphorylation was abolished in the presence of the IGF-I receptor tyrosine kinase inhibitor AG 1024, 2MeSADP did not significantly increase receptor phosphorylation. Nevertheless, phosphorylation of a 120 kDa IGF-I receptor-associated protein was observed. The latter protein was identified by MALDI-TOF/TOF-MS as the proline-rich tyrosine kinase 2 (Pyk2) that co-operates with Src in a PLD2-dependent manner. Consistent with the signalling towards Rap1 and PKB, activation of Pyk2 was abrogated by Ca2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y12 and IGF-I receptors that proceeds through Gβγ-, Ca2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.
Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymati... more Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5Ј-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2Ј-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2Ј-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N 3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2Ј-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products. *Abbreviations: PGE, phenyl glycidyl ether; CZE ES-MS, capillary zone electrophoresis-electrospray mass spectrometry; SPE, solid phase extraction.
ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside level... more ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside levels. The setup comprises a boronate affinity column as a trapping device, a hydrophilic interaction chromatography (HILIC) separation and information-dependent MS detection modes. The system was optimized using standards and tested on biological samples, detecting a number of modified nucleosides. Other urinary biomarkers could also be analyzed by the system developed: for example, the urinary nucleobases were also available for analysis. A simultaneous creatinine-monitoring experiment was also demonstrated to be viable when utilizing the method, which is of benefit as creatinine is a urinary normalizing factor.
The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2&amp... more The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2'-deoxynucleotides and DNA. The modified DNA was hydrolyzed enzymatically, and the mixtures consisting of unmodified and modified 2'-deoxynucleotide adducts were ...
Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods ... more Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods used in their study have utilized low-pressure phenylboronate affinity chromatography materials for the purification of the cisdiol-containing nucleosides. In this study, a boronate HPLC column was surprisingly shown not to trap the nucleosides as would be expected from experience with the classic Affigel 601 resin but showed only partial selectivity toward cis-diol groups while other groups exhibited better retention. In aprotic conditions, trapping of nucleosides was possible; however, the selectivity toward cis-diol-containing compounds was lost with the Lewis basicity of available nitrogens being the main determinant of retention. The experimental findings are compared to and confirmed by DFT calculations.
Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods ... more Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods used in their study have utilized low-pressure phenylboronate affinity chromatography materials for the purification of the cisdiol-containing nucleosides. In this study, a boronate HPLC column was surprisingly shown not to trap the nucleosides as would be expected from experience with the classic Affigel 601 resin but showed only partial selectivity toward cis-diol groups while other groups exhibited better retention. In aprotic conditions, trapping of nucleosides was possible; however, the selectivity toward cis-diol-containing compounds was lost with the Lewis basicity of available nitrogens being the main determinant of retention. The experimental findings are compared to and confirmed by DFT calculations.
ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts fo... more ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts formed between 2′—deoxyadenosine, thymidine, 2′—deoxycytidine and 2′—deoxyguanosine with phenylglycidyl ether is described.
ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts fo... more ABSTRACT The HPLC separation and structure elucidation (UV, NMR, MS) of the nucleoside adducts formed between 2′—deoxyadenosine, thymidine, 2′—deoxycytidine and 2′—deoxyguanosine with phenylglycidyl ether is described.
Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-M... more Using two-dimensional gel electrophoresis (2-DE) and electrospray-tandem mass spectrometry (ESI-MS/MS), we have started the proteome analysis of the cell line Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2). The BY-2 cell suspension culture is widely used as a model system to study the growth and development of plant cells. We present a protocol describing the sample preparation and 2-DE, enabling us to separate and display more than 1000 proteins from this cell culture. A reference gel was generated, using immobilized pH gradient isoelectric focusing in a linear gradient from pH 3 to 10 and 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the tobacco genome is not sequenced yet, a range of protein spots from this reference map was identified by means of a semi-automated liquid chromatography-ESI-quadrupole time of flight-tandem MS (LC-ESI-QTOF-MS-MS) setup and cross-species matching. These data were integrated in a database, which can be accessed at http://tby2-www.uia.ac.be/tby2/. On the on-line reference map, the identified protein spots are hyperlinked to individual protein entries. Each protein entry contains all identification information, as well as links to relevant entries in other on-line databases. Comprehensive search functions are implemented. Especially for an unsequenced but widespread model organism like tobacco BY-2, such a reference database is a convenient source for protein information that brings protein identification within reach without the need for extensive MS. This publicly accessible database provides a solid basis for tobacco BY-2 proteomics in the future.
While protein interaction studies and protein network modeling come to the forefront, the isolati... more While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN-PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well-studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co-IP. Furthermore practical issues concerning the use of this 2-D BN/SDS-PAGE display method for the analysis of protein-protein interactions are discussed.
Journal of the American Society for Mass Spectrometry, 2006
In-source collision induced dissociation was applied to access second generation ions of protonat... more In-source collision induced dissociation was applied to access second generation ions of protonated guanosine. The in-source gas-phase behavior of [BH 2 ] ϩ -NH 3 (m/z 135, C 5 H 3 N 4 O ϩ ) was investigated. Adduct formation and reactions with available solvent molecules (H 2 O and CH 3 OH) were demonstrated. Several addition/elimination sequences were observed for this particular ion and solvent molecules. Dissociation pathways for the newly formed ions were developed using a QqTOF mass spectrometer, permitting the assignment of elemental compositions of all product ions produced. Reaction schemes were suggested arising from the ring-opened intermediate of the protonated base moiety [BH 2 ] ϩ , obtained from fragmentation of guanosine. The mass spectral data revealed that the in-source CH 3 OH-reaction product underwent more complex fragmentations than the comparable ion following reaction with H 2 O. A rearrangement and a parallel radical dissociation pathway were discerned. Apart from the mass spectrometric evidence, the fragmentation schemes are supported by density functional theory calculations, in which the reaction of the ring-opened protonated guanine intermediate with CH 3 OH and a number of subsequent fragmentations were elaborated. Additionally, an in-source transition from the ring-opened intermediate of protonated guanine to the ring-opened intermediate of protonated xanthine was suggested. For comparison, a low-energy collision induced dissociation study of xanthosine was performed. Its dissociation pathways agreed with our assumption
Journal of the American Society for Mass Spectrometry, 2006
no. 11 was inadvertently dropped from the Reference section. The publisher apologizes for this er... more no. 11 was inadvertently dropped from the Reference section. The publisher apologizes for this error. The entry for [11] is for Gaussian 03, and should have been provided as below: 11. Gaussian 03, Revision C.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; and Pople, J. A.; Gaussian, Inc., Wallingford CT, 2004.
Journal of the American Society for Mass Spectrometry, 2005
An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source c... more An in-depth study of the fragmentation pathway of guanosine was conducted by using an in-source collision-induced dissociation high-mass accuracy tandem mass spectrometry experiment. The equivalent of MS 4 data, a level of information normally achieved on ion trap instruments, was obtained on a Q-TOF mass spectrometer. The combination of the features of high-resolution, accuracy, and in-source CID permitted the unambiguous elucidation of the different fragmentation pathways. Furthermore the elemental compositions of the product ions generated were assigned and their mutual genealogical relationships established. Formerly proposed dissociation pathways of guanosine were revisited and elaborated on more deeply. Furthermore, the presence of H 2 O in the collision cell of several tandem MS instruments was demonstrated and its effect on the product ion spectra investigated. The neutral gain of H 2 O by particular fragments of guanosine was experimentally proven by using argon, saturated with H 2 18 O, as the collision gas. Data indicating the occurrence of more complex reactions in the collision cell as a result of the presence of H 2 O were produced, specifically relating to neutral gain/neutral loss sequences. In silico calculations supported the experimental observation of neutral gain by guanosine fragments and predicted a similar behavior for adenosine. The latter was subsequently experimentally confirmed. (J Am Soc Mass Spectrom 2005, 16, 1291-1304 N ucleosides have been established as molecules of exceptional importance in biochemical research for a long period. They are the building blocks of the macromolecules RNA and DNA, exogeneous nucleoside analogs are used as anti(retro)viral and anticancer agents [1], and nucleoside levels in urine have the potential to act as cancer biomarkers . As their occurrence is both ubiquitous and quantitatively varying [6, 7] their unequivocal characterization is crucial.
A study of the interaction of phosphorylated organic compounds with the stainless components of a... more A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography-electrospray ionisation-mass spectrometry system (LC-ESI-MS) was carried out to disclose a (forgotten?) likely pitfall in the LC-ESI-MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC-ESI-MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25-50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.
Abstract 2′-Deoxycitidine (dCyd) and 2′-deoxyguanosine (dGuo) were subjected to reaction with phe... more Abstract 2′-Deoxycitidine (dCyd) and 2′-deoxyguanosine (dGuo) were subjected to reaction with phenylglycidyl ether (PGE) in methanol in order to study the formation of the corresponding 2′-deoxynucleoside adducts. Separation methods were developed on ...
Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum(I1) complex in order to ... more Calf thymus DNA was incubated in vitro with a new aminocoumarin platinum(I1) complex in order to study its interaction with DNA. The platinated DNA was hydrolyzed enzymatically to the 5'-mononucleotide level using DNAase I and nuclease P1. Analysis of the DNA hydrolysate with capillary zone electrophoresis (CZE), using sample stacking, revealed the presence of unhydrolyzed oligonucleotides in the platinated DNA. A homemade system, using only some plastic pipet tips, was constructed to collect the oligonucleotide fraction during CZE analysis. The platinum content of this fraction was determined using graphite furnace atomic absorption with Zeeman background correction. This system proved to be a useful tool to detect platinated DNA species (with a quantifiable detection limit for the detection of platinum of 0.78 ng). Subsequent gel filtration experiments confirmed the presence of high molecular weight oligonucleotides that were platinated. This was proven by reversal of the platination using thiourea and subsequent enzymatic hydrolysis to 5'-mononucleotides.
Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 gli... more Previously it was shown that stimulation of the P2Y12 receptor activates PKB signalling in C6 glioma cells [K. Van Kolen and H. Slegers, J. Neurochem. 89, 442.]. In the present study, the mechanisms involved in this response were further elucidated. In cells transfected with the Gβγ-scavenger β-ARK1/GRK2 or Rap1GAPII, stimulation with 2MeSADP failed to enhance PKB phosphorylation demonstrating that the signalling proceeds through Gβγ-subunits and Rap1. Moreover, Rap1-GTP pull-down assays revealed that P2Y12 receptor stimulation induced a rapid activation of Rap1. Treatment of cells with the Ca2+ chelator BAPTA-AM and inhibition of Src and PLD2 with PP2 or 1-butanol, respectively, abrogated P2Y12 receptor-mediated activation of Rap1 and PKB. In addition inhibition of PKCζ decreased basal and 2MeSADP-stimulated phosphorylation of PKB indicating a role for this PKC isoform in PKB signalling. Although the increased PKB phosphorylation was abolished in the presence of the IGF-I receptor tyrosine kinase inhibitor AG 1024, 2MeSADP did not significantly increase receptor phosphorylation. Nevertheless, phosphorylation of a 120 kDa IGF-I receptor-associated protein was observed. The latter protein was identified by MALDI-TOF/TOF-MS as the proline-rich tyrosine kinase 2 (Pyk2) that co-operates with Src in a PLD2-dependent manner. Consistent with the signalling towards Rap1 and PKB, activation of Pyk2 was abrogated by Ca2+ chelation, inhibition of PLD2 and IGF-I receptor tyrosine kinase activity. In conclusion, the data reveal a novel type of cross-talk between P2Y12 and IGF-I receptors that proceeds through Gβγ-, Ca2+-and PLD2-dependent activation of the Pyk2/Src pathway resulting in GTP-loading of Rap1 required for an increased PKB phosphorylation.
Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymati... more Calf thymus DNA was reacted in vitro with phenyl glycidyl ether (PGE) and was hydrolysed enzymatically, to the 5Ј-monophosphate nucleotides using deoxyribonuclease I (DNA-ase I) and nuclease P1. The adducts were concentrated using solid phase extraction (SPE), on a polystyrene divinylbenzene copolymer in order to remove the unmodified nucleotides. The adducts could be identified using capillary zone electrophoresis-electrospray tandem mass spectrometry (CZE ES-MS/MS), using sample stacking. In addition to the base alkylated 2Ј-deoxynucleotides present in the DNA-hydrolysate, also phosphate alkylated 2Ј-deoxynucleotide adducts were identified for TMP and dAMP. An additional adduct, dUMP alkylated on the uridine moiety was found originating from the hydrolytic deamination of dCMP alkylated on N 3 of the cytosine moiety. Enzymatic hydrolysis using nuclease P1 was incomplete as shown by the presence of dinucleotides alkylated on the base moiety. They were successfully hydrolysed to the corresponding 2Ј-deoxynucleotides by snake venom phosphodiesterase (SVP). Data are shown indicating that alkylations on the pyrimidine bases were more resistant to enzymatic hydrolysis with nuclease P1 than the purine alkylated products. *Abbreviations: PGE, phenyl glycidyl ether; CZE ES-MS, capillary zone electrophoresis-electrospray mass spectrometry; SPE, solid phase extraction.
ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside level... more ESI-MS method is described for targeted metabolomic analysis of urinary modified nucleoside levels. The setup comprises a boronate affinity column as a trapping device, a hydrophilic interaction chromatography (HILIC) separation and information-dependent MS detection modes. The system was optimized using standards and tested on biological samples, detecting a number of modified nucleosides. Other urinary biomarkers could also be analyzed by the system developed: for example, the urinary nucleobases were also available for analysis. A simultaneous creatinine-monitoring experiment was also demonstrated to be viable when utilizing the method, which is of benefit as creatinine is a urinary normalizing factor.
The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2&amp... more The in vitro adduct formation with phenyl glycidyl ethers (PGEs) was studied on 2'-deoxynucleotides and DNA. The modified DNA was hydrolyzed enzymatically, and the mixtures consisting of unmodified and modified 2'-deoxynucleotide adducts were ...
Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods ... more Urinary modified nucleosides have a potential role as cancer biomarkers, and most of the methods used in their study have utilized low-pressure phenylboronate affinity chromatography materials for the purification of the cisdiol-containing nucleosides. In this study, a boronate HPLC column was surprisingly shown not to trap the nucleosides as would be expected from experience with the classic Affigel 601 resin but showed only partial selectivity toward cis-diol groups while other groups exhibited better retention. In aprotic conditions, trapping of nucleosides was possible; however, the selectivity toward cis-diol-containing compounds was lost with the Lewis basicity of available nitrogens being the main determinant of retention. The experimental findings are compared to and confirmed by DFT calculations.
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Papers by Eddy Esmans