14

Biological

ADVERTISING SUPPLEMENT

THE APPLICATION NOTEBOOK FEBRUARY 2006

Identification of Urinary Nucleosides by ESI-TOF-MS

Matthias Pelzing, Thomas Zey, and Gabriela Zurek
Bruker Daltonik GmbH, Bremen, Germany

Dino Bullinger, Antje Frickenschmidt, S. Laufer, and Bernd Kammerer

University of Tuebingen, Institute of Pharmacy, Tuebingen, Germany

The novel method describes the value of true isotopic pattern in conjunction with accurate mass for structural assignment of known and unknown nucleosides from urine samples.

NA, particulary t-RNA, contains a number of modified nucleosides in addition to the regular ribonucleosides adenosine, guanosine, uridine and cytidine. Modifications like methylation, hydroxylation, S/O-substitution and formation of complex side chains are taking place post-transcriptionally within the polynucleotide molecule by various modifying enzyme systems such as methyltranferases and ligases. During RNA turnover, free normal and modified nucleosides are formed by hydrolytic action of ribonucleases and phosphate-elimination by phosphatases. In contrast to unmodified nucleosides, many modified ones are biochemical end products and cannot be reutilized for RNA salvage pathway. Together with small amounts of regular ribonucleosides, the modified species are circulating in the blood stream prior to urinary excretion. Especially modified nucleosides are elevated in cancer patients urine because of the up-regulated metabolism and cell growth in tumour tissue and thus increased RNA turnover. These observations have formed the basis for further investigations of nucleosides as potential tumour markers in cancer diagnosis. The micrOTOF ESI-TOF has proven an indispensable tool for the identification of unknowns based on accurate mass data and the true isotopic pattern. Novel TOF-technology has lead to the conservation of isotope ratios and overcome the past dynamic range limit of this technology. This is especially important for the identification from biological matrices as shown in the example for nucleosides from urine. The analytical pathway is depicted in Figure 1 and described in detail in the experimental section. Experimental A sensitive method for the analysis of urinary ribonucleosides including affinity chromatography purification (AffiGel601, BioRad, Munich, Germany) prior to LCMS analysis using a micrOTOF ESIoa-TOF-MS system (Bruker Daltonik, Bremen, Germany) has been developed. Reversed-phase chromatographic separation of the prepurified nucleosides was performed using an Agilent 1100 binary gradient pump HPLC system with photodiode array detection (Agilent Technologies, Waldbronn, Germany) and a XTerra C18 column (100 2 mm, 3 m particles; Waters). Data acquisition was controlled by Compass 1.0 for micrOTOF.

The mass spectrometer was operated in ESI positive mode in a scan range from 501100 m/z as calibration standard sodium formate solution was injected within the void volume of each chromatographic run. Data evaluation was performed using the Generate Molecular Formula (GMF) software suite within DataAnalysis 3.2 evaluating both accurate mass position and true isotopic patterns for the calculation of molecular formulae.

Figure 1: Analysis pathway for urinary nucleosides.

15 Category ADVERTISING SUPPLEMENT THE APPLICATION NOTEBOOK FEBRUARY 2006

ADVERTISING SUPPLEMENT

Biological

15

Figure 2: In-source fragmentation, neutral loss of m/z 132 for guanosine.

Figure 3: GMF-dialogue with molecular formula proposals for 1-methylguanosine.

Table I: GMF and SciFinder Search Results light blue: new nucleosides in urine; dark blue: confirmation of known nucleoside. No. Measured mass (M H) 1 2 3 4 5 6 7 8 9 10 11 12 346.1259 298.1154 328.1067 384.1171 296.1358 259.0925 286.1036 247.0926 413.1437 282.1190 312.1309 283.1041 Generated molecular ppm formula (M H) C13H20N3O8 C11H16N5O5 C12H18N5O5S C14H18N5O8 C12H18N5O4 C10H15N2O6 C11H16N3O6 C9H15N2O6 C15H21N6O8 C11H16N5O4 C12H18N5O5 C11H15N4O5 error 3.930 2.685 2.009 5.374 1.473 0.329 1.003 0.651 5.173 0.752 2.190 1.537 Sigma value 0.0038 0.0556 0.0475 0.0039 0.0044 0.0072 0.0042 0.0026 0.0064 0.0051 0.0086 0.0068 3-(3-amino-3-carboxy-propyl)-uridine 7-methylguanosine 2-methylthio-N6-methyladenosine N6-succinyladenosine 1,N6-dimethyladenosine 1- -D-ribofuranosyl-1H-imidazole-4-acetic acid N4-acetylcytidine dihydrouridine N6-carbamoylthreonyladenosine 1-methyladenosine N2,N2-dimethylguanosine 1-methylinosine SciFinder search result

Results Affinity chromatography: The affinity chromatography is based on the selective and reversible binding of cis-diols to phenylboronic acid immobilized on a polyacrylamide stationary phase. Elution is performed using 0.1 M formic acid in MeOH/H2O 1:1 (v/v). Chromatographic separation: A mixture of 20 known nucleosides (concentration from 0.8 to 640 nmol/mL based on urine sample values) as well as purified real urine samples were separated with reversed-phase chromatography. UV detection at 260 nm can be applied as additional detection system in case of an aromatic base in the nucleoside. The analysis of real samples clearly demonstrated that the urine samples contain more than the 20 nucleosides from the standard

mixture. Characteristic neutral loss: The characteristic decay of the nucleosides into a neutral sugar moiety (m/z 132) and the corresponding nucleic base fragment is observed under collision induced dissociation (CID) conditions. Figure 2 depicts the mass spectrum of guanosine including the neutral loss. The neutral loss of 132 m/z represents a second independent identification criterion for nucleosides besides affinity chromatography. Furthermore, 132-neutral loss of the selected compounds was confirmed by MSn-experiments (ESI-HCT-MS, tandem mass spectrometry). Generate molecular formula (GMF): Elemental compositions of nucleosides were suggested using the Generate Molecular Formula (GMF)-editor which evaluates the formula suggestion using both accurate molecular mass and isotopic pattern match. To improve

16

Biological

ADVERTISING SUPPLEMENT

ADVERTISING SUPPLEMENT THE APPLICATION

Category 16 NOTEBOOK FEBRUARY 2006

confidence in the results, the measured isotopic pattern is compared with the theoretical one, generated from the suggested elemental composition (sigma value). The smaller the sigma value the better the fit. Figure 3 shows the molecular formula calculated from the mass spectrum of 1-methylguanosine. GMF and database search results from urine sample: Table I contains a selection of identified nucleosides from urine including the mass error and sigma value. Based on molecular formula suggestion and chemical information, a database search (SciFinder) was performed resulting in different structure proposals. The search was restricted to ribosyl-containing structures considering other known fragmentation moieties such as carboxylic acid. The mass spectrometric results obtained by ESI-oa-TOF-MS measurements were compared with our results (characteristic fragmentation patterns) from LC-ESI ion trap MS, tandem mass spectrometry and PSDMALDI-TOF-MS. Five yet unknown nucleosides in urine could be verified (Table I, No. 15). Furthermore, urinary compounds of different origin, for

example, histamine-metabolites (No. 6), could easily be distinguished from RNA-metabolites. Confidence into the presented method was proven by identifying modified urinary nucleosides (No. 712) known to be present in urine. Conclusions A sensitive method for the analysis of urinary nucleosides from urines has been successfully developed and applied to real samples. This proof-of-concept study shows that it is possible to generate structural hypotheses for urinary nucleosides based on accurate mass and true isotopic patterns in combination with database searchs. References
(1) E. Dudley and R.P. Newton, Rapid Comm. in Mass Spectrometry, 14 12001207 (2000). (2) J. Bergmark and G. Granerus, Scand. J. Clin. Lab. Invest., 34 365373 (1974). (3) B. Kammerer and A. Frickenschmidt, JASMS, 16(6) 940947.(2005).

Bruker Daltonics, Inc.

40 Manning Road, Manning Park, Billerica, MA 01821 tel. (978) 663-3660, fax (978) 667-5993 ms-sales@bdal.com, www.bdal.com

For More Information Circle 4