Mass Spectrometric and Spectrophotometric Analyses Reveal an

Jul 8, 2015 - Using a combination of “top-down” and “bottom-up” approaches, and on the basis of state-of-the-art electrospray ionization mass ...
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Mass Spectrometric and Spectrophotometric Analyses Reveal an Alternative Structure and a New Formation Mechanism for Melanin Yuanjiao Li,† Jingjing Liu,‡ Yajie Wang,§ Ho Wai Chan,† Lianrong Wang,*,§ and Wan Chan*,†,‡ †

Department of Chemistry and ‡Environmental Science Programs, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China § Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China S Supporting Information *

ABSTRACT: In this study, we investigated the formation mechanism and chemical structure of melanin that results from the self-assembly of L-3,4-dihydroxyphenylalanine (L-DOPA). Using a combination of “top-down” and “bottom-up” approaches, and on the basis of state-of-the-art electrospray ionization mass spectrometry (ESI-MS) results, we propose a new formation mechanism and an alternative structure for melanin. Specifically, our study of the self-aggregation of L-DOPA based on L-DOPA clusters revealed that melanin is comprised partially of noncovalent supramolecular aggregate that is formed by self-aggregation of L-DOPA and with the individual monomers linked together by a combination of hydrogen bonds, π−π stacking, and ionic bonds. Furthermore, our study showed that unmodified L-DOPA may be part of the building block for melanin in addition to the previously proposed indole derivative based on L-DOPA cyclization. A similar self-aggregation phenomenon was also observed in other structurally related catecholamines, for example, adrenaline.

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Scheme 1. Chemical Structure of L-DOPA and L-DOPADerived Chemical Species That Were Thought To Be Involved in Melanin Formation, Together with the Previously Proposed Chemical Structure for Eumelanin

elanins, a group of naturally occurring dark pigments that are found in most organisms, are produced by the self-assembly of oxidized tyrosine.1,2 Naturally occurring melanins include eumelanin, pheomelanin, and neuromelanin, with eumelanin being the most commonly found.3−5 Melanin plays many critical roles and is crucial for the survival of various organisms.6 For instance, melanin is an effective light absorber that reduces the risk of skin cancer in humans.7,8 Other functional properties of melanin include antioxidative effects and free-radical scavenging.9,10 Despite the importance of melanin for animal survival and its widespread abundance, little is known about its structure and biosynthesis. The chemical properties of melanin, for example, its insolubility in commonly used solvents and resistance to crystallization,11 have hampered the accurate determination of its structure; however, melanin biosynthesis has until now been thought to involve the tyrosinase-catalyzed oxidation of tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) (1, Scheme 1), followed by the self-polymerization of L-DOPA derivatives to form melanin.12 Specifically, L-DOPA has been proposed to undergo further oxidation and cyclization to form indole quinone and dihydroxyindole; the resulting 5,6dihydroxyindole (2) and 5,6-dihydroxyindole-2-carboxylic acid (3) are the main building blocks for eumelanin (4).8,13−15 The goal of this study was to elucidate the chemical structure and biosynthesis of eumelanin under physiological conditions. We initiated melanin formation using a “bottom-up” approach by incubating L-DOPA, the reaction intermediate for melanin biosynthesis, and observed aggregate formation using electrospray © XXXX American Chemical Society

ionization-quadrupole/time-of-flight mass spectrometry (ESIQ-Tof MS) and UV−vis absorption spectrophotometry. The “top-down” approach of analyzing the monomer units dissociated from Sepia off icinalis melanin was also adopted to validate the proposed structure of melanin.

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EXPERIMENTAL SECTION Materials. All chemicals and reagents were of the highest purity available and were used without further purification unless noted otherwise. Acetaldehyde, adrenaline, L-DOPA, L-DOPA-(phenyl-d3), and Sepia of f icinalis melanin were purchased from Sigma (St. Louis). Deionized water was further Received: May 16, 2015 Accepted: July 5, 2015

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DOI: 10.1021/acs.analchem.5b01837 Anal. Chem. XXXX, XXX, XXX−XXX

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quadrupole time-of-flight mass spectrometer (Q-Tof MS) for high-accuracy MS and MS/MS analyses. Self-aggregation of Chemically Modified L-DOPA. L-DOPA was chemically modified with acetaldehyde by adding an excess of acidified acetaldehyde to L-DOPA. After reaction overnight at room temperature, the solution was adjusted to pH 8.5 using an ammonia solution and then allowed to selfaggregate for 30 min before infusion into a Q-Tof MS for highaccuracy MS and MS/MS analyses. ESI-MS and MS/MS Analyses of L-DOPA Aggregates. L-DOPA aggregates were subjected to high-accuracy MS and MS/MS analyses using a Xevo G2 Q-Tof mass spectrometer equipped with a standard ESI interface (Waters, Milford, MA). The capillary and sampling cone voltages for the positive ESI-MS analysis were set at 2.5 kV and 12 V, respectively. The collision energy for the MS/MS experiment was set at 10 eV. The desolvation and ion source temperatures were set at 250 and 70 °C, respectively. ESI-MS and MS/MS Analyses of Adrenaline Aggregates. A structurally related analogue of L-DOPA, that is, adrenaline, was also allowed to self-aggregate using the experimental conditions described above. The self-aggregation of adrenaline was also investigated by analyzing the clusters from the adrenaline self-aggregation using the above-described ESI-MS and MS/MS method. UPLC-MS/MS Analyses of L-DOPA in Purified Melanin. Melanin from Sepia off icinalis was dissolved in 1% hydrogen peroxide (0.1 mg/mL), after which the solution was centrifuged and the supernatant analyzed by UPLC-MS/MS (UPMC, ultraperformance liquid chromatography). Chromatographic

purified using a Milli-Q water purification system (Millipore, Billerica, MA) for use in all experiments. Self-aggregation of L-DOPA. L-DOPA (1 mg/mL) was dissolved in ammonium acetate buffer (50 mM, pH 8.5) and allowed to aggregate at ambient temperature for 30 min before direct infusion (2 μL/min) of the reaction mixture into a hybrid

Figure 1. UV−vis absorption spectrophotometric analysis of L-DOPA self-assembly. L-DOPA (1 mg/mL) was dissolved in ammonium acetate buffer (50 mM, pH 8.6) and allowed to self-aggregate at room temperature. The reaction mixture was analyzed by UV−vis absorption spectrophotometry at various reaction times. Photographic images of the L-DOPA reaction mixture at various reaction times are shown in the inset.

Figure 2. ESI-MS analysis of the L-DOPA self-aggregation reaction mixture. L-DOPA (1 mg/mL) was dissolved in ammonium acetate buffer (50 mM, pH 8.6) and allowed to react for 10 min before direct infusion into an ESI-Q-Tof MS for (A) HR-MS and (B) MS/MS analyses. a, b, c, d, e, and f represent triply charged ion signals assigned to 22-, 23-, 25-, 26-, 28-, and 29-mers, respectively. B

DOI: 10.1021/acs.analchem.5b01837 Anal. Chem. XXXX, XXX, XXX−XXX

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Table 1. High-Accuracy Mass Spectrometric Analysis of Oligomers Resulting from the Self-aggregation of L-DOPA and Acetaldehyde-Modified L-DOPA DOPA

L-DOPA

+ acetaldehyde

measured m/z value

charge (z value)

no. of DOPA units

measured mass, Da

theoretical mass, Da

mass error, ppm

395.1452 592.2134 789.2833 887.8186 986.3534 1084.8906 1183.4263 1281.9508 1380.4945 1479.0247 1577.5574 1676.0945 1774.5967 1873.1320 1511.8612 1643.2357 1708.9286 1840.3615 1905.9745 447.1749 670.2603 893.3451 1004.8920 1116.4310 1227.9648 1339.5148 1451.0605 1562.5914 1674.1271 1413.8638 1488.2163

1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 1 1 2 2 2 2 2 2 2 2 3 3

2 3 8 9 10 11 12 13 14 15 16 17 18 19 23 25 26 28 29 2 3 8 9 10 11 12 13 14 15 19 20

394.1373 591.2055 1576.5508 1773.6164 1970.6910 2167.7654 2364.8368 2561.8858 2758.9732 2956.0336 3153.0990 3350.1732 3547.1776 3744.2482 4532.5599 4926.6834 5123.7621 5517.8808 5714.8998 446.1670 669.2524 1784.6744 2007.7682 2230.8462 2453.9138 2677.0138 2900.1052 3123.1670 3346.2384 4238.5677 4461.6253

394.1376 591.2064 1576.5505 1773.6193 1970.6881 2167.7569 2364.8257 2561.8945 2758.9633 2956.0321 3153.1009 3350.1697 3547.2385 3744.3074 4532.5826 4926.7202 5123.7621 5517.9266 5714.9954 446.1689 669.2534 1784.6757 2007.7601 2230.8446 2453.9290 2677.0135 2900.0980 3123.1824 3346.2669 4238.6047 4461.6892

−0.8 −1.6 0.2 −1.6 1.0 4.0 4.7 −3.4 3.6 0.5 −0.6 1.0 −17.2 −15.8 −5.0 −7.5 −5.3 −8.3 −16.7 −4.3 −1.5 −0.7 4.0 0.7 −6.2 0.1 2.5 −5.0 −9.0 −8.7 −14.3

separation was performed on a Waters HSS C18 column (100 × 2.1 mm, 1.8 μm) eluted at a flow rate of 0.4 mL/min at 40 °C. Gradient elution of 99% A (0.4% formic acid in water) and 1% B (acetonitrile) that increased linearly to 100% B in 5 min was used. The UPLC was coupled to an AB Sciex 4500 QTRAP mass spectrometer operated in the multiple-reaction-monitoring (MRM) mode. TurboIonSpray parameters for positive ion mode ESI-MS were optimized as follows: ion spray voltage, 4000 V; declustering potential, 50 V; entrance potential, 10 V. The collision energy for collision-induced dissociation was set at 20 V. The mass spectrometer for L-DOPA analysis was operated in the MRM mode with the following m/z transitions: 198 → 181; 198 → 152; 198 → 139. The dwell time for each transition was set at 50 ms.

As expected, the spectrophotometric analysis of the L-DOPA reaction mixture revealed a time-dependent increase in absorbance in both the UV and visible regions of the electromagnetic spectrum (Figure 1). As the L-DOPA aggregates grow in size, the increased molecular size of the clusters causes the energy gap between the bonding and antibonding orbitals to decrease. Therefore, the absorbance generally increases as the self-assembly process proceeds and produces melanin of an intense dark color (Figure 1). The corresponding formation of black granules with strong UV absorbing properties is believed to protect humans from UV-induced DNA damage. Analysis of L-DOPA Aggregates by ESI-Q-Tof MS. ESI-MS, which combines the use of a soft ionization technique and sensitive MS detection, is a powerful tool that can be used to investigate the assembly of oligomers as well as aggregates.18,19 To investigate the mechanism of melanin formation, L-DOPA was dissolved in ammonium acetate buffer (50 mM, pH 8.6) and allowed to self-aggregate at room temperature for 30 min. After centrifugation at 10 000 rpm for 5 min, the reaction mixture was infused directly into an ESI-Q-Tof MS (in positive ion mode) to analyze the L-DOPA oligomers. The MS spectrum revealed that L-DOPA and dimeric aggregates were the predominant ions, together with ions representing a wide range of oligomeric clusters (Figure 2). Analysis of the high-accuracy MS results revealed that subsequent L-DOPA clusters differed from each other by an m/z value of 98.5 (Figure 2). Because the ion signals for individual

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RESULTS AND DICSUSSION Study of the Self-aggregation of L-DOPA by Spectrophotometry. L-DOPA resulting from tyrosine metabolism undergoes self-assembly to form melanin, which exhibits a wide range of physiological properties. For instance, melanin protects humans from skin cancer by absorbing DNA-damaging electromagnetic radiation.16,17 The amount of conjugation increases as the self-assembly of L-DOPA proceeds; thus, a red shift in the electromagnetic spectrum is anticipated as L-DOPA aggregates into larger clusters. Therefore, the spectrophotometric changes in L-DOPA aggregates were monitored during polymerization. C

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Scheme 2. Proposed Chemical Structure of Melanin, Together with the Structure of the Supramolecular Aggregate That Is Formed by Self-assembly Using Structurally Related Catecholamines

Figure 3. ESI-MS analysis of the acetaldehyde-modified L-DOPA self-aggregation reaction mixture. Acetaldehyde-modified L-DOPA (1 mg/mL) was dissolved in ammonium acetate buffer (50 mM, pH 8.6) and allowed to react for 10 min before direct infusion into an ESI-Q-Tof MS for (A) HR-MS and (B) MS/MS analyses. a, b, c, d, e, and f represent triply charged ion signals assigned to 22-, 23-, 25-, 26-, 28-, and 29-mers, respectively.

aggregates represent doubly charged ions (z = 2), these results indicate that succeeding clusters differed from each other by a molecular mass of 197.0 Da. This observed mass difference suggests that L-DOPA (1, C9H11NO4, MW = 197.1), together with the previously proposed 5,6-dihydroxyindole (2, C8H7NO2, MW = 149.0) or 5,6-dihydroxyindole-2-carboxylic acid (3, C9H7NO4, MW = 193.0), is the building block for eumelanin.

The individual L-DOPA aggregates were well-characterized by the accurate mass analysis, and the theoretical m/z values were in excellent agreement with the measured m/z values (Table 1). The excellent agreement between the measured and theoretical m/z values of the individual aggregate suggests that unmodified L-DOPA is the repeating unit of eumelanin and that eumelanin is a supramolecule that results from L-DOPA self-aggregation. D

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Figure 4. UPLC-MS/MS analysis of (A) authentic L-DOPA and (B) L-DOPA in Sepia of f icinalis melanin. L-DOPA was eluted at 1.1 min under the developed chromatographic method.

significantly to the observed clustering phenomenon and that the aggregates were formed in the aqueous solution. The proposal that L-3,4-dihydroxyphenylalanine (1) is part of the building block for eumelanin is further supported by the MS/MS analysis. Collision-induced dissociation MS/MS analyses of the L-DOPA trimeric aggregate (m/z 592) revealed fragment ions of the L-DOPA dimeric aggregate (m/z 395) and protonated L-DOPA (m/z 198) to be the predominant ions (Figure 2); this finding unambiguously demonstrates that L-DOPA aggregation yields supramolecular aggregates. A similar fragmentation pattern was also observed when the higher-order aggregate (decameric aggregate) was analyzed using MS/MS (Figure S3). To corroborate the proposed melanin structure, we reacted L-DOPA with acetaldehyde before the aggregation reaction, thus converting L-DOPA to an imine in which the amino group is masked by an ethylidene group (CH3−CH, 6). The ESI-MS analysis of the acetaldehyde-tagged L-DOPA reaction mixture revealed a similar oligomeric pattern (7) to that observed for unmodified L-DOPA (Figure 3), although the molecular mass of the repeating unit was 26 Da larger than that observed for unmodified L-DOPA aggregation. The structures of the oligomeric aggregates were well-characterized based on accurate MS (Table 1) and MS/MS analyses (Figure 3). The observation that the spectrum of acetaldehyde-modified L-DOPA oligomeric aggregate (Figure 3A) was similar to that of aggregated unmodified L-DOPA (Figure 4A) confirms that unmodified L-DOPA (1), in addition to the previously proposed

The results from the above ESI-MS analysis revealed that no small molecules (e.g., water or hydrogen) are eliminated as L-DOPA undergoes self-aggregation. Therefore, self-aggregation of L-DOPA may occur in melanin biosynthesis and melanin may partially be a noncovalent supramolecular aggregate. In addition to the previously proposed covalent mechanism, melanin synthesis might occur through self-aggregation, with the monomer units linked together by a combination of hydrogen bonds, π−π stacking, and ionic bonds (5, Scheme 2).20 Similar noncovalently bonded amino acid oligomeric aggregates have been observed in previous studies using positive ESIMS.21,22 It has been suggested that the presence of extensive intermolecular hydrogen bonds can stabilize aggregates and allow their existence in an aqueous environment.22 For example, a high abundance of serine octameric clusters was observed by Cooks et al. in ESI-MS analyses.22 Using a combination of collision-induced dissociation MS/MS and density functional calculations, the clustering phenomenon was attributed to the strong hydrogen-bonding networks that formed between neighboring carboxylic acid groups and in between the hydroxyl and amino groups.22 The potential of ESI to promote clustering was also investigated. To this end, we varied the analyte concentration (1.0, 0.2, and 0.1 mg/mL; Figure S1) and the capillary spray voltage (3.5, 2.5, and 1.0 kV; Figure S2) to examine their effects on the cluster distribution. However, neither experiment revealed any significant changes in the cluster pattern (Figures S1 and S2). These results indicated that the ESI did not contribute E

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ACKNOWLEDGMENTS W. Chan thanks the Hong Kong University of Science and Technology for Startup Funding (Grant R9310). We also express our sincere gratitude to Prof. Zhong-Ping Yao (Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University) for helpful discussions.

cyclized indole forms (2, 3), are the building blocks for eumelanin. We therefore propose that eumelanin is a partially noncovalently supramolecular aggregate (5). A similar noncovalent self-aggregation mechanism has previously been proposed for the synthesis of polydopamine through dopamine self-aggregation.23,24 To investigate the existence of the proposed phenomenon in other structurally related catecholamines, we have also analyzed the reaction mixtures from self-aggregation of adrenaline (8, Scheme 2). ESI-MS analysis revealed a similar spectrum of a supramolecular aggregate of adrenaline (9, Figure S4) as that observed for L-DOPA (5). These studies reaffirmed our proposal that a combination of hydrogen bonds, π−π stacking, and ionic bonds binds the L-DOPA together, forming eumelanin. UPLC-QqQ MS Analysis of L-DOPA in Purified Melanin. To validate our proposal, we also explored the feasibility of identifying L-DOPA in melanin isolated from Sepia off icinalis. Upon partial dissolution of the melanin in 1% hydrogen peroxide, the existence of L-DOPA in the solution was analyzed by UPLC-QqQ MS (QqQ MS, triple-quadrupole mass spectrometry) of high sensitivity and selectivity. UPLC-QqQ MS analysis of the melanin solution showed unambiguous identification of L-DOPA resulting from the dissolution of L-DOPA from melanin (Figure 4). Results from this study are in excellent agreement with our proposal that melanin is partially a supramolecular aggregate of L-DOPA held together by noncovalent interactions (5).

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ASSOCIATED CONTENT

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REFERENCES

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Using state-of-the-art analytical techniques, we investigated in this study the chemical structure and formation chemistry of the not well-understood melanin. Upon analyzing L-DOPA and its structurally related chemical analogs, we proposed a new formation mechanism and an alternative structure for melanin. Our study revealed noncovalent aggregation of L-DOPA, together with the previously proposed covalently bonded structures, is the structure of melanin. It is expected that this discovery will provide new directions/insights investigating the biological functions of melanins.

* Supporting Information S

Effect of changing analyte concentration and the capillary spray voltage in the cluster distribution. ESI-MS/MS analysis of the L-DOPA decamer; ESI-MS analysis of the adrenaline and L-DOPA-(phenyl-d3) self-aggregation reaction mixture; and ESI-MS/MS analysis of L-DOPA together with the cleavage reactions for the formation of major fragment ions. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01837.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: (852) 2358-7370. Fax: (852) 2358-1594. E-mail: [email protected]. *Phone: (86) 027-68753391. Fax: (86) 027-68753391. E-mail: [email protected]. Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acs.analchem.5b01837 Anal. Chem. XXXX, XXX, XXX−XXX