Quantification of Amino Acid Enantiomers Using Electrospray Ionization and Ultraviolet Photodissociation

The enantioselectivity of tryptophan (Trp) for amino acids, such as alanine (Ala), valine (Val), and serine (Ser), was investigated using ultraviolet (UV) photoexcitation and tandem mass spectrometry. Product ion spectra of cold gas-phase amino acid enantiomers that were hydrogen-bonded to Na+(L-Trp) were measured using a variable-wavelength UV laser and a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. Na+(L-Trp), formed via amino acid detachment, and the elimination of CO2 from the clusters were observed in the product ion spectra. For photoexcitation at 265 nm, the relative abundance of Na+(L-Trp) compared to that of the precursor ion observed in the product ion spectrum of heterochiral Na+(L-Trp)(D-Ala) was larger than that observed in the product ion spectrum of homochiral Na+(L-Trp)(L-Ala). A difference between the Val enantiomers in the relative abundance of the precursor and product ions was observed in the case of photoexcitation at 272 nm. The elimination of CO2 was not observed for L-Ser for the 285 nm photoexcitation, which was the main reaction pathway for D-Ser. Photoexcited Trp chiral recognition was applied to identify and quantify the amino acid enantiomers in solution. Ala, Val, and Ser enantiomers in solution were quantified from their relative abundances in single product ion spectra measured using photoexcitation at 265, 272, and 285 nm, respectively, for hydrogen-bonded Trp within the clusters.

INTRODUCTION e identi cation of amino acid enantiomers is critical in the eld of biochemistry, because D-amino acids play signi cant roles in living organisms. 1,2) Amino acid enantiomers are distinguished using analytical methods based on chromatography, nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. 3) Mass spectrometry is widely used for analyses of molecular structures because it is highly sensitive, selective, and suitable for analyzing mixtures and for detecting amino acid mutations in peptides. 4,5) e identi cation of amino acid enantiomers is also critical in the eld of mass spectrometry, and several mass spectrometry-based techniques for this have been developed over the past two decades. 6,7) Collision-activated dissociation and ion mobility-mass spectrometry of gas-phase, copper-bound complexes is used to distinguish amino acid enantiomers by detecting variations in enantiomer binding energies and collision cross-sections, respectively. 8,9) NMR spectroscopy of gas-phase ions using a magnetic resonance acceleration technique has also been proposed. 10) e chiral recognition of biomolecules is attributed to the homochirality in biomolecules consisting of L-amino acids and D-sugars. e origin of biomolecular homochirality in living organisms is clearly one of the most scienti cally important issues of our time. e extraterrestrial origin of enantiomeric excess in amino acids has been evaluated in numerous studies on abiotic amino acid syntheses in simulated interstellar ice, [11][12][13] amino acid analyses of meteorites, 14,15) and enantioselective destruction by circularly polarized light. 16,17) e remarkable homochiral preference of gas-phase serine (Ser) octamers has been investigated using mass spectrometry-based techniques. [18][19][20][21] Gas-phase proline clusters also exhibited a homochiral preference. 22,23) e physical and chemical properties of cold gas-phase hydrogen-bonded clusters have been investigated as a model for chemical evolution in interstellar molecular clouds because chemical reactions in space occur at low temperatures and densities. 24) D-Tryptophan (D-Trp), when photoexcited at 266 nm, under-goes dissociation via CO 2 loss when it was noncovalently complexed with L-Ser or L-threonine (L-r) in the presence of Na + , whereas such an enantioselective reaction was not observed for the noncovalent complex with alanine (Ala). 25) is indicates that the side-chain OH group of the molecule contributed to the enantioselective photodissociation.
For hydrophilic amino acids such as Ser, r, glutamine, asparagine, glutamic acid, and aspartic acid, enantioselective dissociation induced by 266 nm photoexcitation was observed in the product ion spectra of hydrogen-bonded clusters with Na + (L-Trp). 25,26) In the case of a hydrophobic amino acid such as Ala, enantioselective dissociation was not observed and its enantiomers could not be discriminated. 25) is is one of the limitations in the chiral discrimination of amino acids when photoexcitation at 266 nm is used, which was the fourth harmonic of a compact and inexpensive Nd:YAG laser.
In this study, amino acid enantiomer selectivity was investigated using the ultraviolet (UV) photoexcitation of Trp in cold gas-phase hydrogen-bonded clusters containing hydrophobic amino acids, such as Ala and valine (Val), in addition to Ser, a hydrophilic amino acid. Hydrogen-bonded Trp chiral recognition in cold gas-phase clusters generated via electrospray ionization and collisional cooling was used in the identi cation and quanti cation of amino acid enantiomers in solution.

EXPERIMENTAL
Product ion spectra of hydrogen-bonded clusters of amino acid enantiomers were measured using a wavelength-variable UV laser and a home-built tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. 27) Hydrogen-bonded clusters of amino acid enantiomers with Na + (L-Trp) were generated via the electrospray ionization of 1.0 mM solutions that contained L-Trp, NaCl, and analyte molecules in a mixture of water and methanol. e ionization source was operated at a sample ow rate of 3 µL/min, a sheath gas ow rate of 3 L/ min, and an applied voltage of 2 kV. e hydrogen-bonded cluster ions were transferred to the gas phase through a metal capillary and octopole ion guide. e gas-phase ions were mass-selected using a quadrupole mass lter and thermalized in a cold ion trap (8 K) via multiple collisions with He bu er gas. e mass-selected, temperature-controlled ions were then irradiated with a photoexcitation laser pulse at 1.2 mJ/pulse, which was unfocused so as to allow spatial overlap between the ion packets and laser pulse. e product ion masses were determined using a re ectron time-ofight spectrometer. e ion signals were quanti ed using dual microchannel plates (F4655, Hamamatsu Photonics, Hamamatsu, Japan) and a digital storage oscilloscope (104MXi, Teledyne LeCroy, Chestnut Ridge, NY, USA). e photoexcitation laser pulse was the third harmonic of a tunable Ti:sapphire laser (LT-2211T, LOTIS TII, Minsk, Belarus) pumped by the second harmonic of a Nd:YAG laser (LS-2145TF, LOTIS TII).

UV photoexcitation of hydrogen-bonded clusters
Product ion spectra measured using 265 nm photoexcitation at 8 K of Ala, Val, or Ser enantiomers hydrogen-bonded with Na + (L-Trp) are shown in Fig. 1. Na + (L-Trp), formed via amino acid detachment, and the loss of CO 2 from the clusters were observed. e loss of CO 2 from non-zwitterionic amino acids with NH 2 and COOH groups was reported during UV photoexcitation studies of an ice matrix. 28) e zwitterionic Trp with NH 3 + and COO − groups dissociated via the loss of NH 3 . 29) erefore, the product ion spectra shown in Fig. 1 indicate that the amino acids within the clusters are non-zwitterionic structures that contain NH 2 and COOH groups. e relative abundance of Na + (L-Trp) (m/z 227) in the product ion spectrum of heterochiral Na + (L-Trp)(D-Ala) (m/z 316) is larger than that in the product ion spectrum of homochiral Na + (L-Trp)(L-Ala).
e product ion spectra of heterochiral Na + (D-Trp)(L-Ala) and homochiral Na + (D-Trp) (D-Ala) were identical with the spectra of heterochiral Na + (L-Trp)(D-Ala) and homochiral Na + (L-Trp)(L-Ala), respectively. Ala enantiomers are identi ed by the relative photodissociation cross sections of the cold gas-phase hydrogen-bonded clusters. In contrast, no di erences between Val enantiomers were observed in the product ion spectra, as shown in Figs. 1c and d. In the Ser product ion spectra, the relative abundance of the CO 2 -eliminated ion (m/z 288) compared to that of Na + (L-Trp) for heterochiral Na + (L-Trp)(D-Ser) (m/z 332) is larger than that for homochiral Na + (L-Trp)(L-Ser). Ser enantiomers are identi ed by the photoinduced elimination of CO 2 , as previously reported. 25) erefore, di erent enantioselective phenomena are observed within cold gas-phase hydrogen-bonded clusters containing Ala, Val, or Ser.

Photoexcitation wavelength and enantioselectivity
To investigate the e ect of photoexcitation wavelength on the enantioselectivities, UV photoexcitation studies were conducted on the cold gas-phase hydrogen-bonded clusters in the wavelength range of the ππ * state of the Trp indole ring. e photoexcitation wavelengths used were 265, 272, and 285 nm. It has been reported that photoexcitation at 272 and 285 nm can be used to distinguish isomeric amino acids and pentoses, respectively. 30,31) In the case where the cold gas-phase hydrogen-bonded clusters were photoexcited at 272 and 285 nm, Na + (L-Trp), formed via amino acid detachment, and the loss of CO 2 from the clusters were observed in the product ion spectra as in the case of photoexcitation at 265 nm (Fig. 1). e dependence of photoexcitation wavelength on the photodissociation of the Ala, Val, and Ser enantiomers are displayed in Fig.  2 with the D-and L-amino acids plotted using closed and open circles, respectively. e relative abundance ratio R 1 is derived by dividing the abundance of the Na + (L-Trp) by the abundance of the precursor ion in the product ion spectrum of the hydrogen-bonded cluster with Na + (L-Trp).
As shown in Fig. 2a, the di erence in R 1 values between the Ala enantiomers observed in the product ion spectra of photoexcitation at 265 nm (Figs. 1a and b) decreases at 272 and 285 nm. In contrast, the di erence in R 1 values between Val enantiomers is largest for photoexcitation at 272 nm. Hence, photoexcitations at 265 nm and 272 nm are optimal for distinguishing Ala and Val enantiomers, respectively.
Ser enantiomers display the same tendency in the photoexcitation-wavelength dependence of R 1 , as displayed in Fig. 2c. To examine the e ects of the photoexcitation wavelength on the enantioselective reaction, the relative abundance ratios R 2 for Ala, Val, and Ser enantiomers were derived by dividing the abundance of the CO 2 -eliminated ion by the abundance of Na + (L-Trp) in the product ion spectrum. e R 2 values are plotted as a function of photoexcitation wavelength in Fig. 3. For the clusters of Ala enantiomers that are hydrogen-bonded with Na + (L-Trp), Na + (L-Trp), formed via the detachment of Ala, and the elimination of CO 2 from the clusters can be observed in the product ion spectra in the case of photoexcitation at 265, 272, and 285 nm. In addition, R 2 does not depend on the photoexcitation wavelength. For Val enantiomers, CO 2 elimination from the clusters is not observed in the product ion spectra for the 285 nm photoexcitation, which is the main reaction pathway in the case of photoexcitation at 265 and 272 nm. However, no di erences in R 2 values were observed between Val enantiomers at these wavelengths. e enantiomers in the hydrogen-bonded clusters containing Ala or Val exhibited the same photoreactivity and wavelength dependence, thus making them indistinguishable using R 2 . e relative abundance of the 265 nm photoinduced CO 2 elimination for D-Ser was larger than that of the 265 nm photoinduced CO 2 elimination for L-Ser, as shown in Figs. 1e, 1f, and 3c.  In contrast, no CO 2 elimination was observed for L-Ser at photoexcitation of 285 nm, which is the main pathway for D-Ser. us, the enantioselectivities and photoexcitationwavelength dependences are di erent for Ala, Val, and Ser.
Wavelength dependence of enantioselective reactions was reported for cold gas-phase hydrogen-bonded clusters of Trp and carbohydrate enantiomers. e enantioselective reactions observed at 265-280 nm were caused by a photoinduced electron transfer from the indole ring to the carboxyl group of Trp. 32) e ππ * state of the Trp indole ring observed at 285 nm contributed to the recognition of chiral pentose units. 31) For the chiral recognition between amino acids reported here, the hydrogen-bonding and electronic structures of Trp within the amino acid clusters may be critical, as in the case of the carbohydrates. A knowledge of the geometric and electronic structures of the cold gasphase hydrogen-bonded clusters is therefore crucial for developing an understanding of the molecular recognition mechanism responsible, which could be further studied using photodissociation spectroscopy, ion mobility spectrometry, and theoretical calculations.

Quanti cation of amino acid enantiomers in solution
Ala and Val enantiomers were identi ed by the relative photodissociation cross sections in the case of photoexcitation at 265 and 272 nm, respectively, whereas Ser enantiomers could be identi ed by photoinduced CO 2 elimination. Hydrogen-bonded Trp chiral recognition was applied to the quanti cation of amino acid enantiomers in solution via the UV photoexcitation of cold gas-phase hydrogen-bonded clusters with Na + (L-Trp) generated via electrospray ionization and collisional cooling.
To construct a calibration curve for quantifying the Ala enantiomer in solution, the product ion spectra at photoexcitation of the hydrogen-bonded clusters at 265 nm were measured for several L-and D-Ala enantiomeric mixtures, with L-Ala to D-Ala mole fractions of 1.00, 0.75, 0.50, 0.25, and 0.00. e 1.00 mol fraction of L-Ala to D-Ala represents an L-Ala solution containing L-Trp, with the product ion spectrum of Na + (L-Trp)(L-Ala) displayed in Fig. 1b. e 0.00 mol fraction of L-Ala to D-Ala represents a D-Ala solution containing L-Trp, with the product ion spectrum of Na + (L-Trp)(D-Ala) displayed in Fig. 1a. From the product ion spectra at photoexcitation of the mixtures at 265 nm, the ratio of the relative abundance R 1 is derived by dividing the abundance of Na + (L-Trp) by the abundance of the precursor ion, and the natural logarithm of R 1 is plotted as a function of the mole fraction of L-Ala to D-Ala in Fig. 4. A linear relationship is observed with an intercept of −2.069, a slope of −0.921, and a correlation coe cient (r 2 ) of 0.989. For Val enantiomers that were examined using photoexcitation of 272 nm, the intercept, slope, and r 2 of the linear relationship are −2.264, 0.469, and 0.987, respectively, as shown in Fig. 5. erefore, the mole fraction of L-enantiomer in solution was determined from R 1 derived from the single product ion spectrum. e key dissociation parameters, the excitation energy and temperature of the gas-phase ions, were controlled using a single-nanosecond UV laser pulse and a cold ion trap, respectively. 33) However, the relative abundance of the product and precursor ions, R 1 , is dependent on the laser power and the spatial overlap between the ion packet and the laser pulse. us, the linear R 1 relationship should be calibrated with both the L-and D-enantiomers for each experimental condition. e relative abundance of product ions formed via onephoton absorption, R 2 , is independent of the laser power and the spatial overlap between the ion packet and the laser pulse. Ser enantiomers are identi ed using R 2 , as shown in Fig. 3c. To construct a calibration curve for quantifying the Ser enantiomer using 285 nm photoexcitation, the product ion spectra of L-and D-Ser mixtures were obtained, as for Ala and Val. e natural logarithm of R 2 is plotted as a function of the L-Ser to D-Ser mole fraction in Fig.  6, where R 2 is derived by dividing the abundance of the CO 2 -eliminated ion by the abundance of Na + (L-Trp) in the product ion spectrum. A linear relationship for photoexcitation at 285 nm was observed. erefore, photoexcited Trp recognizes amino acid enantiomers through intermolecular hydrogen bonds.

CONCLUSION
Enantiomer selectivity among amino acids was investigated using UV photoexcitation of cold gas-phase hydrogen-bonded clusters generated via electrospray ionization and collisional cooling. Amino acid enantiomers were identi ed based on optical properties and enantioselective reactions of hydrogen-bonded Trp within the clusters. Ala, Val, and Ser enantiomers in solution were quanti ed from their relative abundances in single product ion spectra that were obtained by the photoexcitation of hydrogen-bonded Trp within the clusters at 265, 272, and 285 nm, respectively. Enantioselective phenomena of amino acids were observed in studies regarding chemical evolution in interstellar molecular clouds. Insights into the molecular characteristics and their origins regarding chemical evolution would be valuable for analytical method development. Fig. 6. Linear plot of ln(R 2 ) for Na + (L-Trp)(Ser) at 8 K vs. the mole fraction of L-Ser to D-Ser in solution. e relative abundance ratio R 2 is derived by dividing the abundance of the CO 2eliminated ion by the abundance of Na + (L-Trp) in the product ion spectrum measured by photoexcitation at 285 nm at 8 K for Na + (L-Trp)(Ser).