A study of kynurenine fragmentation using electrospray tandem mass spectrometry

A Study of Kynurenine Fragmentation usingElectrospray Tandem Mass Spectrometry

Santiago Vazquez and Roger J. W. TruscottDepartment of Chemistry, Australian Cataract Research Foundation, University of Wollongong, NSW,Australia

Richard A. J. O’HairSchool of Chemistry, The University of Melbourne, Victoria, Australia

Allan WeimannDepartment of Clinical Pharmacology, Rigshospitalet, Copenhagen, Denmark

Margaret M. SheilDepartment of Chemistry, University of Wollongong, NSW, Australia

A combination of accurate mass measurement and tandem mass spectrometry (both production and precursor ion scans) have been used to characterize the major fragment ions observedin the ESI mass spectrum of kynurenine. Kynurenine is a metabolite of tryptophan found in thehuman lens and is thought to play a role in protecting the retina from UV-induced damage.Three major fragmentation pathways were evident, following initial elimination either ofammonia, H2O and CO or the imine form of glycine. The latter is proposed to occur via theformation of an ion-molecule complex. In the case of loss of H2O and CO from deaminatedkynurenine, there is evidence for an acylium ion intermediate, which is not observed for theloss of H2O and CO directly from protonated kynurenine. Product ion scans of deuteratedkynurenine enabled the elucidation of structural rearrangements that were not evident in thespectra of the native compound. Since UV filter compounds can often only be isolated in smallquantities from the lens, this study forms the basis for the characterization of novel UV filtercompounds using mass spectrometry. The approach presented here may also be useful for thecharacterization of related classes of small molecules. (J Am Soc Mass Spectrom 2001, 12,786–794) © 2001 American Society for Mass Spectrometry

Kynurenine (Kyn) is found in the human lensalong with related tryptophan metabolites in-cluding 3-hydroxykynurenine (3OHKyn), 3-

hydroxykynurenine-O-glucoside (3OHKG) and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside(AHBG) [1, 2]. These compounds are thought to play arole in protecting the lens and retina from UV-inducedphotodamage and in reducing chromatic aberration,thus sharpening the image on the retina [2]. Along withtheir presence in the lens, these tryptophan metaboliteshave been found to be associated with other patholog-ical conditions. 3OHKyn in particular, has been foundat significantly elevated levels in the brains of patientssuffering from dementia associated with human immu-nodeficiency virus (HIV) infection, hepatic encephalop-athy, Parkinson’s disease and Huntington’s disease

[3–5]. In neuronal cell cultures, 3OHKyn has beenfound to be cytotoxic at concentrations as low as 1mM[6]. Recently, it has been suggested that these trypto-phan metabolites may also be implicated in the forma-tion of age-related cataract [7, 8], however, their role isnot fully understood and is the subject of on-goingresearch [9].

One difficulty with the characterization of UV filtercompounds and their derivatives is that often they areonly isolated from the lens in very small quantities, thusprecluding facile characterization by nuclear magneticresonance (NMR) spectroscopy. Therefore, electrospraymass spectrometry, which has inherently higher sensi-tivity than NMR, is invaluable for the structural char-acterization of new UV filter compounds. For example,the utility of mass spectrometry in the characterizationof a novel fluorophore glutathionyl-3OH-kynurenine-glucoside (GSH-3OHKG) isolated from the human lenswas demonstrated recently [10].

There have been a number of reports in which massspectrometry has been used for the analysis of Kyn and

Published online May 8, 2001Address reprint requests to Professor Margaret Sheil, Department ofChemistry, University of Wollongong, NSW 2522, Australia. E-mail:[email protected]

© 2001 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received September 12, 20001044-0305/01/$20.00 Revised February 17, 2001PII S1044-0305(01)00255-0 Accepted February 28, 2001

derivatives in biological samples [11–19]. The onlyreport of the fragmentation mechanisms of Kyn andderivatives, however, used electron ionization (EI) andtherefore involved fragmentation of odd-electron ionswith relative high internal energies [20]. The presentwork describes a detailed study of the fragmentation ofKyn observed in electrospray (ESI) and tandem massspectra using a variety of techniques including: precur-sor ion scans [21, 22]; accurate mass measurements; anddeuterium exchange [23–27] in combination with themore commonly-used product ion scans [21, 23]. Thiswork will form a basis for future study of other Kynderivatives and ultimately, for the application of thisapproach to the identification of novel Kyn derivativesisolated from the human lens. The multi-faceted ap-proach employed here may also be a useful model forelectrospray tandem mass spectrometry studies of otherclasses of amino acids and small molecules.

Experimental

Formic acid and acetonitrile (HPLC grade) were pur-chased from Ajax (Australia). Kynurenine was obtainedfrom Sigma Chemical Co. (St. Louis, MO, USA). D2Oand DCl (purity 99.9%) were obtained from CambridgeIsotope Laboratories (Andover, MA). All gases usedwere compressed high purity obtained from BOC gases(Chatswood, NSW, Australia). Milli Q water was usedto prepare all solutions.

ESI mass spectra, precursor ion scans, and production scans (for breakdown graphs) were recorded on aVG Quattro (VG Biotech, Altrincham, Cheshire, U.K.)which has a quadrupole, hexapole, quadrupole config-uration in which each quadrupole has an m/z range of4000. All spectra were acquired in positive ion mode atunit mass resolution using multichannel analysis dataacquisition. The instrument has been upgraded to meetthe Quattro II specifications. The instrument conditionsused were: electrospray probe tip voltage 3.5 kV; 0.5 kVon chicane counter electrode; cone voltage 25–70 volts;nitrogen was used for both the bath and nebulizing gasflowing at 350 L/h and 10 L/h respectively; scan rate100 m/z per second and typically 10–20 scans weresummed to obtain representative spectra. The solvent(1% formic acid in 50% aqueous acetonitrile) was deliv-ered using a Harvard Apparatus model 22 syringepump (South Natick, MA, USA) operating at a flow rateof 10 mL/min. Kyn was dissolved in 50% aqueousacetonitrile (containing 1% formic acid) to give a con-centration of 100 pmol/mL and 10 mL were injected foreach analysis. The source temperature was set at 85 °C.Both MS-1 and MS-2 were calibrated using a solution ofsodium iodide.

Tandem mass spectrometry (MS/MS) experimentson the VG Quattro were carried out in positive ionmode only, using the same conditions described above.For product ion scans of the non-deuterated species theresolution of MS-1 was set to a minimum allowing thepassage of 2–3 Da, while in precursor ion scans it was

set to allow unit mass resolution. In both cases thecollision cell was filled with Ar gas at a pressure of3.5 3 1024 mbar and the collision energy was variedbetween 0 and 90 eV to achieve the desired level offragmentation.

Accurate mass measurements and tandem mass spec-trometry studies of deuterated Kyn were conducted onan Autospec oa-TOF magnetic sector mass spectrometer(VG Analytical Ltd. Wythenshawe, UK) equipped withan electrospray ion source. The mass spectrometer has amagnetic sector of EBE configuration as MS-1 and anorthogonal acceleration time-of-flight analyzer as MS-2,i.e., EBE-TOF. The voltage on the electrospray probe tip(ca. 8 kV) was optimized for individual samples toobtain the most stable and intense signal. Nitrogen wasused as the nebulizing gas (concurrent to the solvent)and a counter current flow of warm nitrogen (80 °C, 1atm) was employed to assist with solvent evaporation.

Tandem mass spectrometry experiments on the EBE-TOF were carried out only in positive ion mode. Meth-ane was used as the collision gas and the pressure in thecollision cell was adjusted during the course of each runto maximize the diagnostic fragmentation obtained foreach experiment. The voltage applied to the collisioncell was 400 V. All tandem mass spectra were acquiredover an m/z range of 50–250. Tandem mass spectra ofproduct ions of interest were obtained by generatingthese ions via collision-induced dissociation (CID) inthe source using raised cone and skimmer voltages. Theions of interest were then selected as the precursor ionin MS-1 and subsequently passed through the collisioncell after which the MS/MS spectrum was recorded byMS-2.

Accurate mass measurements were performed bysetting the resolution in the magnetic sector analyzer to10,000 (at 10% valley peak-to-peak) and then scanningthe accelerating voltage to span a range of m/z 6 20% ofthe ion of interest. Calibration of the instrument wasachieved through the use of amino acid standards,which had m/z values within the m/z 6 20% range forthe particular product ion analyzed.

Deuterated Kyn, for labeling experiments, was pro-duced by dissolving solid Kyn in D2O with 0.01 M DCl,to facilitate both the exchange and ionization process.This solution (concentration of 100 pmol/mL) was theninfused into the mass spectrometer a rate of 10 mL/min.

Results and Discussion

Electrospray Ionization Mass Spectrum ofKynurenine

The ESI mass spectrum of Kyn is shown in Figure 1,with the structure of Kyn inset. The spectrum shows theprotonated molecular ion [M1H]1 at m/z 209 and seriesions that have been identified by accurate mass mea-surements and tandem mass spectrometry to be derivedfrom Kyn. The relative intensity of these product ionsincreased with increasing cone voltage (data not

787J Am Soc Mass Spectrom 2001, 12, 786–794 FRAGMENTATION OF KYNURENINE

shown) indicating that they are formed predominantlyvia collision-induced dissociation in the intermediatepressure region of the source. Focusing effects in thisregion, which may cause differences in ion intensity onaltering the cone voltage, are not likely to be significantover this small mass range [28].

Table 1 summarizes the accurate mass measure-ments of the major ions in the ESI mass spectrum(Fig. 1). In all but one case, the measured masses ofthe product ions are within 65 ppm of those expectedfrom the proposed composition. The exception is theion occurring at m/z 174 with a difference of 7.4 ppm.We remain confident of the proposed composition,however, since other possible compositions for thision would have had a mass difference of .50 ppm.Further, these assignments are supported by theresults of tandem mass spectrometry experiments (dis-cussed later).

Tandem Mass Spectrometry

The product ion spectrum of the protonated molecularion of Kyn, m/z 209 (not shown), yielded the same majorfragment ions as the ESI mass spectrum, albeit therelative intensity of each varied with collision energy.Figure 2 shows the relative intensity of the majorproduct ions versus laboratory collision energy (Elab).The most abundant product ion at low collision ener-gies was the deaminated fragment at m/z 192. Therelatively high abundance of this ion, even at the lowestcollision energies, demonstrated that loss of ammoniafrom the protonated molecular ion was a particularlyfacile process. As the collision energy increased, how-ever, the intensity of this ion decreased and was accom-panied by an increase in intensity of the ions at m/z 74,118, 120 and 136. The relative intensity of m/z 136, 118and 74 reached maxima at Elab values of approximately

Figure 1. The ESI/MS of protonated Kynurenine. The structureof Kyn is shown in inset. The ion at m/z 74 (not shown) was alsopresent with an abundance of 10.5%.

Table 1. Accurate mass measurements of the major ions observed in the electrospray mass spectrum of protonated Kyn

Observedm/z

Calculatedm/z

Errora

(ppm)

SuggestedElemental

Composition Assignment

209.0935 209.0926 24.3 C10H13N2O3 [M1H]1

192.0663 192.0661 21.0 C10H10NO3 2NH3

174.0542 174.0555 7.4 C10H8NO2 2(NH31H2O)163.0869 163.0871 1.2 C9H11N2O 2(CO1H2O)146.0603 146.0606 2.1 C9H8NO 2(NH3, H2O1CO)136.0767 136.0762 23.7 C8H10NO 2(C2H3NO2)120.0445 120.0449 3.3 C7H6NO 2(H2O1CO, C2H5N)118.0656 118.0657 0.8 C8H8N 2(C2H3NO2, H2O)

aThe error is given as the ppm difference between the calculated and measured m/z.

Figure 2. Relative intensity of product ions versus laboratorycollision energy in the product ion spectra of protonated kynure-nine (m/z 209). Data for the product ions m/z 174 and m/z 163 werenot shown, however, the ions followed the same trend as m/z 146.

788 VAZQUEZ ET AL. J Am Soc Mass Spectrom 2001, 12, 786–794

10, 40, and 55 eV respectively, while the intensity of them/z 120 gradually increased with collision energy acrossthe whole range examined. The product ions m/z 174and 163 are not shown since they closely followed them/z 146 ion (Figure 2).

A series of precursor ions scans were also conductedon the major ions observed in the product ion spectrum.In these experiments MS-2 was set to the m/z of theproduct ion of interest and MS-1 was scanned toidentify all the precursors of that ion. Figure 3 showsthe precursor ion scans for m/z 118, 120, and m/z 146.Precursor ion scans of m/z 118 (Fig. 3c) and m/z 120 (Fig.3b) showed that these ions were formed by the directelimination of H2O or C2H5N from ions at m/z 136 or163, respectively. The precursor ion scan of m/z 146revealed, however, that this product ion had threeprecursor ions. Elimination of H2O and CO, from m/z192, was the major decomposition pathway while theloss of CO and NH3 from the ions at m/z 174 and 163,respectively, were minor decomposition routes asjudged by the relative intensity of the precursor ions.Precursor ion scans were also performed on m/z 136, m/z163, m/z 174 and m/z 192 (data not shown). These dataenabled identification of three major pathways as sum-marized in Scheme 1. The three pathways, shown inScheme 1, each involved initial loss of a small neutralmolecule from the protonated molecular ion, i.e., elim-ination of ammonia, H2O, and CO or glycine in theimine form.

Elimination of ammonia from the protonated molec-ular ion to give the product ion at m/z 192, is the firststep in the principal dissociation route of protonated

Kyn. Dookeran et al. have studied the fragmentation ofa large number of amino acids and found that the initialfragmentation steps of protonated a-amino acids de-pend strongly on the identity of the R group inH2NCH(R)CO2H [29]. Aliphatic amino acids show thesequential elimination of H2O and CO while aromaticamino acids show the loss of NH3 [29]. Since kynure-nine is an aromatic amino acid, our observations are inagreement with that study [29]. The elimination of NH3

from Kyn was also observed as the base peak in the EImass spectrum of Kyn [20].

Following the elimination of ammonia, a product ionis observed at m/z 146, which corresponds to the elim-ination of CO2H2. Chemical ionization (CI) mass spectraof carboxylic acids also show loss of CO2H2 fromprotonated molecular ions as a two step process involv-ing initial loss of H2O to form a stable acylium ion,followed by elimination of CO [30–33]. In contrast,a-amino acids cannot form stable acylium ions via theelimination of H2O [34]. This has been rationalized byTsang and Harrison [35], who concluded that the pos-sible acylium ion formed was higher in energy than itsproduct ions, and may dissociate spontaneously losingCO [35]. It is interesting to note that we observed bothdirect elimination of CO2H2 from the deaminated prod-uct ion at m/z 192 and via a pathway involving theformation of the intermediate acylium ion (more be-low).

The dissociation pathway involving the initial elim-ination of the immonium form of glycine (C2H3O2N) toyield the ion at m/z 136, is proposed to occur via theformation of an ion/molecule complex as shown inScheme 2. The breakdown graph for protonated Kyn(Fig. 2) shows that the loss of NH3 decreases rapidly inimportance as the collision energy is raised. The prod-

Figure 3. Precursor ion scans for the product ions (a) m/z 146, (b)m/z 120, and (c) m/z 118 of protonated kynurenine.

Scheme 1. Fragmentation pathways of protonated Kyn. Thebold arrows indicate major decomposition routes.


uct ion at m/z 136 from loss of the imine initiallyincreases in intensity but then decreases, as the frag-ment ion at m/z 74 becomes the most intense ion. Thisobservation is most readily explained by assuming theformation of an ion molecule complex a. At low internalenergies, proton transfer occurs within the complex toform species b. With increasing internal energy, how-ever, the complex may separate before proton transfercan occur and the immonium ion c is formed. Followingthe formation of the m/z 136 ion, H2O is readily elimi-nated from b to generate a product ion at m/z 118.

The third major process occurring is the loss ofCO2H2 directly from the molecular ion to yield theproduct ion at m/z 163. In this instance, the intermediateacylium ion (from loss of H2O) was not observed, whichis in contrast to the elimination of CO2H2 from thedeaminated fragment (m/z 192). This suggests that theacylium ion formed directly from the protonated mo-lecular ion is not stable and rapidly dissociates to givethe ion at m/z 163. This is characteristic of the fragmen-tation of amino acids as noted above. The m/z 163 ionfurther decomposes through one of two fragmentationpathways: either the loss of C2H5N to give the ion at m/z120 or the loss of NH3 generating the ion at m/z 146.

Tandem Mass Spectra of Deuterated Kynurenine

Although the accurate mass measurements and tandemmass spectra enabled the elemental composition andthe origin of the product ions to be established, defini-tive structures could not be assigned without furtheranalyses. Hence, product and precursor ion scans wereperformed on Kyn that had been labeled by deuteriumexchange to assist in characterization of the majorproduct ions. The ESI tandem mass spectrum of deu-terated Kyn (d6), acquired on the EBE-TOF instrument,is shown in Figure 4. Kyn is particularly suited todeuterium labeling since it has six hydrogen atomsavailable for exchange in the region of the molecule

where fragmentation occurs. There is the possibility ofthe exchange of a further hydrogen atom by deuteriumthrough the enolization of the benzylic ketone, how-ever, the 1H NMR of Kyn in D2O (data not shown) didnot show any exchange occurring in this region. Fur-ther, in work carried out by Bond et al. on the 13C NMRof Kyn over a range of pH no evidence was presentedfor the formation of the enol, which would cause achange in the 13C chemical shift [36]. These precursorion scans, performed on the triple quadrupole instru-ment, enabled the identification of the precursor-prod-uct ion relationships for each of the deuterated productions as shown in Scheme 3. The pathways shown in the

Scheme 2. Proposed mechanism for elimination of the iminefrom protonated Kyn via the formation of an ion moleculecomplex.

Figure 4. Product ion spectrum of deuterated Kyn (d6) obtainedusing the EBE-TOF instrument.

Scheme 3. Fragmentation pathways of deuterated Kyn, deter-mined from product spectra of deuterated Kyn (d6) fragments.


scheme are analogous to those of unlabelled kynure-nine (Scheme 1).

Product ion scans on individual fragment ionsformed via CID in the source also enabled us todetermine the number of exchangeable hydrogens ineach ion, greatly assisting the process of identifyingpossible structures. These experiments were conductedon the EBE-TOF instrument because it enabled higherresolution precursor ion mass selection to ensure therewas no interference from species in which kynureninewas not deuterated to the same degree. The samefragment ions were observed in the ESI mass spectra oneach instrument, although there were some minor dif-ferences in the relative intensity of the ions. Figure 5shows the product ion spectra of the ions at m/z 195 (d3),m/z 167 (d4) and m/z 140 (d4). These are analogous to theions observed at m/z 192, m/z 163 and m/z 136 in the ESImass spectrum of unlabelled kynurenine.

Elimination of Deuterated Ammonia

In one major fragmentation pathway, deuterated Kyneliminates a molecule of ND3 from the side chainresulting in the deaminated fragment ion at m/z 195.The product ion scan of this ion (Figure 5a) showed twopairs of ions for each dissociation step, thus suggestingthe existence of parallel dissociation routes for thedeaminated fragment (m/z 195).

The existence of two dissociation pathways wasinterpreted in terms of the presence of two structuralisomers of the deaminated fragment, the cyclized d anduncyclized e forms (Scheme 4). We suggest that thesestructures direct the form of deuterated water that islost from the deaminated fragment. In ab initio studiesof protonated glycine, for the loss of water to take placethe transfer of exchangeable hydrogen from nitrogen tothe OH must first occur [37, 38]. The prominent loss ofHDO from e suggests the involvement of a source ofexchangeable hydrogen in the structural rearrange-ments, which we suggest to be the two acidic hydro-

gens shown in e. Elimination of D2O from d suggeststhe absence of hydrogen in the exchange process. Weexpect that the likely source of exchangeable deuteriumor hydrogen would be the deuterium of the deuteratedaromatic amine. Following the elimination of D2O andHDO from f and g, carbon monoxide is subsequentlyeliminated to yield j and k, respectively.

The structure of the deaminated product ions f, g aresimilar to those expected for carboxylic acids in the gasphase. In fact it may be expected that these ions wouldact as carboxylic acids during the elimination of water,producing a stable acylium intermediate. In the case ofamino acids, however, the elimination of water pro-duces an a-amino acylium ion that undergoes facileexothermic loss of CO to form immonium ions [39]. Thisrapid two-step decomposition may be additionally fa-cilitated by the ability of the final product ion tostabilize the remaining positive charge. In the case ofcarboxylic acids, however, the acylium intermediate isconsiderably more stable, possibly a consequence of theinability of the product ion to stabilize the chargebeyond the production of the acylium ion.

From product ion spectra of the fragment ions f andg it was apparent that the overall [M1H1–H2O–CO]1

decomposition process proceeded via parallel routes(Scheme 4). Elimination of the two neutral speciesoccurs without the detection of the acylium intermedi-ate or detection of the intermediates h and i. Theintermediates are sufficiently stable to allow their de-tection by the instrument. While the direct loss of formicacid can not be discounted, given the rationale men-tioned earlier, we suggest that the reason for absence ofthe intermediates in the parallel decomposition routesmay be due to the facilitation of CO elimination bysome means, for example by the ability of final productions, j and k, to better accommodate the charge. We arecurrently investigating whether this is also evident inthe MS/MS spectra of related compounds.

Elimination of Deuterated Glycine as the Imine

The formation of an electrostatically-bound ion mole-cule complex prior to fragmentation (Scheme 2) hasbeen proposed for the elimination of glycine as theimine. This could account for the production of com-plementary fragment ions from, in this case, the deu-terated molecular ion.

At lower collision energies the fragment ion l(Scheme 5) is expected to be the more prominent of thetwo possible ions formed. Further fragmentation of lleads to the elimination of D2O (Figure 5c) resulting inthe formation of an indole type structure, n, with twodeuterium atoms. The formation of this stable cyclicstructure was supported by the fact that additionalproduct ions were observed in the tandem mass spec-trum of m/z 120 (not shown).

In association with the peak owing to n, evident atm/z 120 (Figure 5c), there was another peak one m/zhigher. We proposed the fragment ion o has the same

Figure 5. Product ion spectra of ions from deuterated kynure-nine generated by in-source CID (a) m/z 195, (b) m/z 167, and (c)m/z 140 obtained using the EBE-TOF instrument.


structure as n with the difference in m/z arising from thenumber of deuterium atoms associated with each frag-ment. The additional deuterium atom in structure omay be located on the carbon either a or b to thenitrogen through H/D scrambling resulting from tau-tomerization of l to p and q (Scheme 5). This mechanismmay also account for the peak at m/z 122 in Figure 5(c)that corresponds to the exchange of two deuteriumatoms in the same manner.

At higher collision energies (.20 eV) the a-glycylcation m is expected to be the most prominent of thetwo ions. This species is essentially the immonium formof glycine and the analogous species has been previ-ously observed in the CI mass spectrum of aspartic acid[35]. Further, the tandem mass spectrum of m gave aproduct ion at m/z 29 (1DN§CH) which would beproduced by the elimination of D2O and CO is evident.This is in agreement with results reported by othergroups [40–42]. This ion is also apparent in the disso-ciation of glycine [43].

Elimination of Deuterated Water and CarbonMonoxide

For the pathway involving elimination of D2O and CO,deuterated Kyn displays the dissociation chemistryexpected for a-amino acids [29]. The carboxylic acidgroup fragments through the elimination of D2O andCO without the detection of the acylium intermediate.The apparently spontaneous decomposition of this acy-lium intermediate, the rationale for which has beenpresented above, gave rise to the immonium ion, struc-ture r in Scheme 6. The product ion spectrum of rshowed elimination of the enamine ND2HC¢CH2 orND2HC¢CDH producing s or t. The hydrogen/deute-rium scrambling evident in the proposed structures fors and t suggests a contribution from the aromaticnitrogen-bound deuterium during the elimination pro-cess. This would only be possible if two pathwaysexisted for the elimination of the enamine. Path (a),direct elimination of the enamine from r or path (b),

Scheme 4. Major product ions and proposed structures observed in the product ion spectrum of[MH1 2 ND3]1 m/z 195.


Scheme 5. Major product ions observed in the product ion spectrum of [MH1 2 Imine]1 m/z 140.

Scheme 6. Major product ions observed in the product ion spectrum of [MH1 2 D2O 2 CO]1 m/z 167.


exchange of hydrogen and deuterium between thearomatic amine and the carbon a to the carbonyl groupfollowed by elimination of the triply deuterated enam-ine by a similar mechanism to path (a). Further evi-dence for the structures s and t is provided via thepresence of the expected product ions at m/z 93 and 94(corresponding to the elimination of carbon monoxidefrom the proposed structures) respectively in the tan-dem mass spectra of r and s (not shown).

Conclusions

The present study demonstrates that collisional induceddissociation reactions of protonated kynurenine add toexisting knowledge of the even electron dissociationchemistry of small molecules, and offer a potentiallyeffective means for structural characterization usingtandem mass spectrometry. Although this study onlyexplored the fragmentation of protonated kynurenine,this molecule displays a diverse range of fragmentationroutes and mechanisms. Deuterium exchange experi-ments coupled with product ion scans provided us withthe greatest source of structural information and sec-ondly, revealed the variety of structural rearrangementsthat the fragment ions underwent during CID, whichare not normally observed because of the isobaricnature of the ions formed. These routes cover manyaspects of conventional dissociation chemistry and it isexpected that understanding the fragmentation behav-ior of protonated Kyn will aid in the structural charac-terization of novel UV filter compounds and Kyn de-rivatives.

AcknowledgmentsThe Australian Research Council supported this work. Grantsfrom the Australian Research Council, the Ramaciotti Foundation,and University of Wollongong enabled the purchase of the elec-trospray mass spectrometers. SV is grateful for an AustralianPostgraduate Award; AW is grateful for a Fellowship from theDanish Natural Sciences Research Council.

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Documents

A study of kynurenine fragmentation using electrospray tandem mass spectrometry