B American Society for Mass Spectrometry, 2016

J. Am. Soc. Mass Spectrom. (2016) 27:1227Y1235 DOI: 10.1007/s13361-016-1396-y

RESEARCH ARTICLE

Carbon Dots and 9AA as a Binary Matrix for the Detection of Small Molecules by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Yongli Chen,1 Dan Gao,3,4 Hangrui Bai,3,4 Hongxia Liu,3,4 Shuo Lin,1 Yuyang Jiang2,3 1

Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Graduate School at Shenzhen, Peking University, Shenzhen, 518055, China 2 National and Local United Engineering Laboratory for Personalized Antitumor Drugs, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, China 3 State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, China 4 Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen, 518055, China

Abstract. Application of matrix-assisted laser-desorption/ionization mass spectrometry (MALDI MS) to analyze small molecules have some limitations, due to the inhoCOOH CO OC mogeneous analyte/matrix co-crystallization and interference of matrix-related peaks OH O H in low m/z region. In this work, carbon dots (CDs) were for the first time applied as a - stacking hydrogen-bond interaction binary matrix with 9-Aminoacridine (9AA) in MALDI MS for small molecules analysis. + + Analytes + + By 9AA/CDs assisted desorption/ionization (D/I) process, a wide range of small Analytes COOH HOOC molecules, including nucleosides, amino acids, oligosaccharides, peptides, and antiCOOH HOOC cancer drugs with a higher sensitivity were demonstrated in the positive ion mode. A COOH HOOC detection limit down to 5 fmol was achieved for cytidine. 9AA/CDs matrix also exhibited HOOC excellent reproducibility compared with 9AA matrix. Moreover, by exploring the ionization mechanism of the matrix, the influence factors might be attributed to the four parts: (1) the strong UV absorption of 9AA/CDs due to their π-conjugated network; (2) the carboxyl groups modified on the CDs surface act as protonation sites for proton transfer in positive ion mode; (3) the thin layer crystal of 9AA/CDs could reach a high surface temperature more easily and lower transfer energy for LDI MS; (4) CDs could serve as a matrix additive to suppress 9AA ionization. Furthermore, this matrix was allowed for the analysis of glucose as well as nucleosides in human urine, and the level of cytidine was quantified with a linear range of 0.05–5 mM (R2 > 0.99). Therefore, the 9AA/CDs matrix was proven to be an effective MALDI matrix for the analysis of small molecules with improved sensitivity and reproducibility. This work provides an alternative solution for small molecules detection that can be further used in complex samples analysis. Keywords: 9-Aminoacridine, Carbon dots, Binary matrix, MALDI MS, Small molecules CDs

HO

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Received: 1 February 2016/Revised: 14 March 2016/Accepted: 22 March 2016/Published Online: 13 April 2016

Introduction

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atrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a soft ionization tool developed by Karas and Hillenkamp [1] in 1980s, has become a powerful tool for bioanalysis because of the

Electronic supplementary material The online version of this article (doi:10. 1007/s13361-016-1396-y) contains supplementary material, which is available to authorized users. Correspondence to: Dan Gao; e-mail: [email protected]

advantages of high throughput, high sensitivity, high salt tolerance, and relative ease of sample preparation procedure [2, 3]. However, the analysis of low molecular weight (LMW) compounds (<500 Da) still faces some challenges. The conventional matrices, such as 2, 5-dihydroxybenzoic acid (DHB) and a-cyano-4-hydroxycinnamic acid (CHCA), are not suitable for small molecules detection because of their strong background interferences in the low mass range (m/z 100–500). Moreover, visible Bsweet spot^ is generated during the cocrystallization process of analyte and matrix, which usually leads to poor reproducibility and unreliable quantification results. Therefore, development of new matrices with easy

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preparation, excellent ionization efficiency, and uniform crystallization is highly desirable. Much effort has focused on producing new matrices to enhance the analytical performance for the detection of LMW compounds. One approach is the development of organic matrices with high sensitivity and reduced background, such as 9aminoacridine (9AA) [4], 1,5-diaminonaphthalene [5], isoliquiritigenin [6], and N-(1-naphthyl) ethylenediamine dinitrate [7]. For example, 9AA is an excellent matrix for some small molecules detection in negative ion mode, but the poor solubility usually leads to inhomogeneous crystallization with the analytes, thus decreasing the sensitivity of MALDI MS and shot-to-shot reproducibility [8]. In addition, many inorganic nanoparticles, including porous silicons [9], metal and metal oxide nanoparticles [10–13], as well as carbon-based nanomaterials [14–16] with different compositions and morphology have been explored to be effective MALDI matrices. In particular, carbon-based nanomaterials such as graphite, graphene, fullerene, carbon nanotube, and diamond nanowire have attracted great attention in laser desorption/ionization (D/I) because of their outstanding charge mobility and universal optical absorption properties [16–19]. However, fullerene and fullerene-derivatives showed poor sensitivity for small molecules analysis [20]. In contrast, graphene and carbon nanotube exhibited higher sensitivity, but their poor dispersibility in aqueous solution decreased the spot-to-spot reproducibility, and the ionization efficiency was often affected by their different structural properties [19, 21]. Carbon nanodots (CDs), which is a new form of zero-dimensional carbonaceous nanomaterial, possesses a variety of advantages, including convenient sample synthesis, excellent water dispersibility, and photostability [22, 23]. Recently, Nie’s group firstly used CDs as a novel matrix for the analysis of a series of LMW compounds by MALDI-TOF MS with higher sensitivity over other reported carbon based matrices [24]. However, the intense carbon cluster signals of this matrix were exhibited, which caused matrix suppression effect and complicated mass spectra in the low mass range. Moreover, the application of CDs matrix paid less attention to the further development and in-depth mechanism study in positive or negative ion mode. Therefore, there is still a requirement to improve the analytical performance of the existing MALDI matrices, especially the best organic or inorganic matrices by far. Recently, some binary matrices have been proposed to overcome the above drawbacks of singular matrix and improve the quality of D/I process via suppressing the matrix clusters and fragments [25], producing homogeneous sample-matrix co-crystallization [26], expanding the detection range [27], and increasing the detection sensitivity [28]. However, the binary matrices generated with carbon-based materials have never been proposed with increased sensitivity and reproducibility. Taken into consideration the excellent features of CDs and the advantages of binary matrix, a new binary matrix composed of CDs and 9AA was developed for small molecules detection by MALDI-MS. Similar to the existing reports [29],

Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

our synthesized CDs exhibited nano-sized morphology, excellent water dispersibility and broad UV absorption properties. Importantly, it was observed that the addition of CDs to 9AA solution yielded higher detection signals for the analysis of amino acids, nucleosides, oligosaccharides, peptides, and anticancer drugs. Moreover, 9AA/CDs binary matrix produced a more homogeneous co-crystallization of matrix and analyte that improved the spot-to-spot and shot-to-shot reproducibility to some extent. The new binary matrix was applied for glucose and nucleosides detection, and then extended for cytidine quantification in urine sample. This work demonstrates that 9AA/CDs-assisted LDI MS method enables a simple, rapid, high sensitivity and reproducibility application for LMW compounds detection.

Experimental Chemicals and Reagents Trifluoroacetic acid (TFA), 9-aminoacridine (9AA), D-glutamic acid, D-aspartic acid, D-histidine, D-lysine, DL-methionine, D-proline, L-glutamine, L-asparagine, adenosine, cytidine, thymidine, uridine, and sodium citrate were purchased from Sigma-Aldrich (St Louis, MO, USA). Glucose, lactose, raffinose, doxorubicin, curcumin, paclitaxel, and etoposide were obtained from J&K Scientific Co. Ltd. (Beijing, China). α-Cyano-4-hydroxycinnamic acid (CHCA) was purchased from Bruker Daltonics GmbH (Bremen, Germany). Four peptides, Tyr-Gly-Gly, Tyr-Phe, Phe-Gly-Phe-Gly, and Arg-Ser-GlyPhe-Tyr were purchased from Shanghai Apeptide Co. Ltd. (Shanghai, China). Bond Elut Phenylboronic Acid (PBA) columns were obtained from Varian (Agilent, Santa Clara, CA, USA). Deionized water (18 MΩ) used in all experiments was prepared from a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Synthesis of CDs CDs was synthesized by the hydrothermal strategy [30], which is a simple, convenient one-step method. Briefly, 0.2 g sodium citrate and 1.5 g NH4HCO3 were dissolved in 10 mL water; the mixture was then sealed into a stainless steel autoclave and heated at 180 °C for 4 h. After the reaction completed, the autoclave was cooled down naturally. Next, the aqueous solution was centrifuged at 16,000 rpm for 15 min to dislodge the nonfluorescent deposit and get the upper CDs aqueous solution for use. The CDs was conducted through a dialysis membrane (1000 Da, molecular weight cutoff) for 48 h and then dried under vacuum.

Characterization of CDs High resolution transmission electron microscopy (HRTEM, Tecnai G2 F30; FEI, Hillsboro, OR, USA) was used to characterize the surface morphology of the prepared CDs. Fourier transform infrared spectroscopy (FTIR) spectra were measured

Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

by an EQUINOX 55 (Bruker) spectrometer with the KBr pellet technique ranging from 500 to 4000 cm–1. X-ray photoelectron spectroscopy (XPS) measurements were taken with an Esca Lab 250 spectrometer (Thermo Scientific, Waltham, MA, USA) with monochromatic Al Kα radiation (1486.6 eV). The UV-vis absorption spectra of products were recorded using a Beckman Coulter DU-800 spectrophotometer (Miami, FL, USA) at room temperature in 1.0 cm path length. Fluorescence measurement of CDs was performed with a Fluorolog-3 fluorescence spectrophotometer (Horiba, JobinYvon, France) using a 1.0 cm quartz cell equipped with a thermostatic bath. The samples were excited at 350 nm, and the emission spectral range was from 370 to 650 nm. The emission slit and the exciting slit were both 2 nm.

Sample Preparation Standard solutions of amino acids, nucleosides, oligosaccharides, peptides, and anticancer drugs were prepared by directly dissolving corresponding chemicals in water at a concentration of 10 mmol L–1 as stock solution. The solutions were prepared by mixing and diluting the stock solutions with water to give the concentration of 1 mmol L–1 for each analyte. Urine sample was collected from a healthy female volunteer and mixed with 3-fold amount of methanol for protein precipitation. The precipitated proteins were removed by centrifuging at 16,000 rpm for 30 min. The Bond Elute PBA-SPE column was preconditioned with a mixture of H2O-ACN (70:30, v/v) with 1% formic acid and 100 mmol L–1 ammonium formate buffer (pH 10.0), separately. The sample was then loaded into the column and washed with 100 mmol L–1 ammonium acetate buffer (pH 8.5). Finally, the sample was eluted with H2O-ACN (70:30, v/v) solution containing 1% formic acid and collected for further analysis. For matrix preparation, the CDs solution was obtained in H2O-EtOH (50:50, v/v) at various concentrations of 0.2, 0.5, 1, 2, 5, and 10 mg mL–1. 9AA matrix was prepared with the concentration of 5 mg mL–1 in H2O-EtOH (50:50, v/v). Then, the CDs with the optimum concentration were mixed with an equal volume of 10 mg mL–1 9AA as a binary matrix. CHCA was dissolved in H2O-ACN (30:70, v/v) at a concentration of 20 mg mL –1 containing 0.1% trifluoroacetic acid (TFA), and mixed with an equal volume of 10 mg mL–1 9AA solution. After that, 1 μL of the analyte was premixed with 1 μL of matrix in a centrifuge tube, and then 1 μL of the resulting mixture was pipetted on a 384 AnchorChip target plate (Bruker Daltonics) and air-dried for further MS analysis.

Mass Spectrometry Analysis A MALDI-tandem-time-of-flight (MALDI-TOF/TOF) mass spectrometer (Bruker Daltonics) equipped with a 337 nm nitrogen laser (10.0 Hz) was used for small molecules detection under reflectron and positive ion modes. Each spectrum was the cumulative average of 1000 laser shots in a pixel with 100 μm in diameter. Ions in the mass range of 0-1000 were detected with a sampling rate of 2.5 GS s-1. The laser power

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energy was adjusted as needed. Besides, MS/MS experiments were also carried out to help identify the potential structures of glucose and nucleosides in urine sample. The MS/MS spectra were acquired in positive LIFT mode in the mass range of 160400 performed with a medium focus. Data were processed using FlexAnalysis 3.3 software (Bruker).

Results and Discussion Characterization of CDs The obtained CDs solution exhibited a long-term homogeneous phase without any noticeable precipitation at room temperature. The TEM image showed that these CDs were mono-dispersion, photochemical stable, and water soluble, and the size distributed in the range from 2 to 8 nm (Supplementary Figure S1a). The HRTEM image revealed high crystallinity of the CDs with the lattice spacing of 0.21 nm, agreed with that of in-plane lattice spacing of graphene (Supplementary Figure S1b) [31]. The FTIR image of CDs was presented in Supplementary Figure S1c, which exhibited characteristic absorption bands of O-H and N-H stretching vibrations at 3450 cm –1 , C-H stretching vibrations at 2964 cm–1, C=O and C-O stretching vibrations at 1732 cm–1 and 1586 cm-1, as well as C-H bending vibrations at 1346 cm–1 and 1224 cm–1. In addition, XPS measurements were taken for the surface elements, and the result showed that carbon and oxygen were the main elements presented at the surface of CDs (Supplementary Figure S1d and Supplementary Figure S2). These data revealed that the as-prepared CDs were functionalized with –COOH and/or –OH groups, which made it possible for liberating and transferring proton to analyte in positive ion MALDI MS. Furthermore, a strong absorption of the CDs in the UV region (250–350 nm) revealed the advantage for absorbing laser energy and transferring energy to analyte (Supplementary Figure S1e) [32]. The fluorescent spectrum indicated the CDs possessed great fluorescence properties. Then, the laser desorption/ionization (LDI) mass spectrum of CDs was described in Supplementary Figure S1f. As expected, the CDs exhibited a clean background in the low mass region (m/z <1000), showing a free matrix effect for the analysis of LMW compounds by MALDI-MS, which was better than the previously reported CDs matrix [24].

Optimization of the Matrix for MALDI MS Analysis In this study, our initial objective was to improve the analytical performance of LDI-MS for LMW compounds detection with the synthesized CDs. For this purpose, lactose (L, MW = 342.30), cytidine (C, MW = 243.22), histidine (His, MW = 155.16), and glutamine (Gln, MW = 146.15) were randomly chosen as model small molecules for the analysis. To our surprise, these molecules exhibited poor sensitivity when using CDs as matrix (Figure 1, shown as black line). This might be due to the different physicochemical properties, including absorption, morphology, and size features

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Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

Figure 1. MALDI-TOF MS spectra of standard solutions containing (a) lactose (m/z 365.311, [M + Na]+; m/z 381.309, [M + K]+), (b) cytidine (m/z 266.218, [M + Na]+; m/z 282.220, [M + K]+), (c) histidine (m/z 178.091, [M + Na]+), and (d) glutamine (m/z 169.122, [M + Na]+; m/z 185.134, [M + K]+) by using CDs (black), 9AA (blue), and 9AA/CDs (red) matrices in positive ion mode. The signal peak at m/z 195 is the [M + H]+ of 9AA matrix. The amount of each analyte is set as 500 pmol. The same laser power of 40% is applied for the analytes. Laser intensity: 40%

when using different synthesis conditions and precursors. In addition, the previous studies indicated that 9AA was a wellknown matrix for LMW compounds detection with reduced matrix background in the mass spectra [8, 33]. Interestingly, as shown in Figure 1 (red line), the signal intensities obtained from 9AA/CDs were much higher than those of 9AA and CDs, and better signal peaks were generated when using positive ion MALDI MS (Supplementary Table S1). Moreover, different concentrations of CDs (0.2, 0.5 1, 2, 5, and 10 mg mL–1) were mixed with an equal volume of 10 mg mL–1 9AA separately to confirm the optimum concentration of this matrix for small molecules detection. The result showed that 1 mg mL–1 CDs obtained the highest signal intensities for the detected molecules and were ultimately used for the following experiments (Figure 2).

the sample surface, which might lead to poor quantification (Figure 3a and b). Nevertheless, as shown in Figure 3c, the

Matrix Crystallization and Signal Reproducibility Evaluation

Figure 2. Effect of CDs concentrations on the small molecules detection using MALDI-TOF MS in reflective positive ion mode; 10 mg mL–1 9AA and CDs in diverse concentrations (0.2, 0.5, 1, 2, 5, and 10 mg mL–1) are mixed as a binary matrix for the analysis of lactose (green), cytidine (purple), glutamine (blue), and histidine (red). The amount of each analyte is set as 500 pmol. Laser intensity: 40%

His was chosen as a model molecule to compare the cocrystalline morphology of matrices with different solvent mixtures. For 9AA/CDs in H2O-EtOH (30:70, v/v) and H2OMeOH (30:70, v/v), irregular crystallizations were formed onto

Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

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Figure 3. The optical images of 9AA/CDs and His co-crystals prepared in (a) H2O-EtOH (30:70, v/v), (b) H2O-MeOH (30:70, v/v), and (c) H2O-EtOH (50:50, v/v). (d) The co-crystalline morphology of 9AA and His prepared in H2O-EtOH (50:50, v/v). The signal distribution of His obtained from (e) 9AA/CDs and (f) 9AA matrices; the spatial resolution is set as 100 μm

crystallization of 9AA/CDs in H2O-EtOH (50:50, v/v) with a small and thin layer of crystal was more homogeneous than that of 9AA/CDs prepared in H2O-EtOH (30:70, v/v) and H2O-MeOH (30:70, v/v). For exploration of the reproducibility, the intensities from 22 acquisitions were stabilized at around 60,000 and the relative standard deviation (RSD) was about 6.8% (Figure 3e). In addition, the signals from different spots also revealed a better reproducibility with RSD of 5.4% (n = 10) by 9AA/CDs matrix (Supplementary Figure S3). The results indicated that the CD/9AA matrix has the capability to increase spot-to-spot and shot-to-shot reproducibility of MALDI MS. Moreover, the crystal of 9AA was distributed irregularly compared with those of 9AA/CDs (Figure 3d), and their spot-to-spot and shot-to-shot intensity reproducibility exhibited with RSD of 27.8% (Figure 3f). Therefore, 9AA/CDs matrix prepared in H2O-EtOH (50:50, v/v) solution was used for the ensuing MALDI MS experiments.

Analysis of the Various Types of LMW Compounds To test the performance of the binary matrix for the analysis of LMW molecules, nucleosides, amino acids, oligosaccharides, peptides, and some anticancer drugs were detected with 9AA, 9AA/CHCA, and 9AA/CDs matrices in positive ion mode. Nucleosides are the RNA metabolites and become the wellknown biomarkers in various diseases. In this study, a nucleosides solution containing adenosine (A, MW = 267.24), cytidine (C, MW = 243.22), guanosine (G, MW = 283.24), and uridine (U, MW = 244.20) were analyzed in positive ion mode. As shown in Figure 4a, when using 9AA as matrix, poor signal intensities and S/N ratios of sodium adducts [M + Na]+ and potassium adducts [M + K] + were shown, and their intensities did not affect after combination with the traditional matrix CHCA. However, when 9AA/CDs was applied, both

the signal intensities and S/N ratios of the majority of nucleosides were improved approximately 4-fold, and cytidine even obtained a limit of detection (LOD) of 5 fmol (Supplementary Figure S4). Interestingly, the signals of the tested nucleosides with Na+ adducts revealed the highest sensitivity, and no any H+ adduct was detected. This might be caused by the introduction of sodium citrate during the CDs synthesis step. Next, an amino acid mixture containing proline (Pro, MW = 115.13), histidine (His, MW = 155.16), glutamic acid (Glu, MW = 147.13), asparagine (Asn, MW = 132.12), and methionine (Met, MW = 149.21) was also analyzed. As expected, the result showed that [M + Na]+ and [M + K]+ of the amino acids were only detected at fairly low intensity, and Met was even unable to be determined by 9AA matrix. 9AA/CHCA matrix showed a little enhancement compared with those of 9AA. In addition, 9AA/CDs matrix exhibited the highest sensitivity with [M + Na]+ and [M + K]+ ions for all the analytes (Figure 4b). Then, a standard peptide mixture containing Tyr-Phe (YF, MW = 328.14), Tyr-Gly-Gly (YGG, MW = 295.12), Phe-Gly-Phe-Gly (FGFG, MW = 426.19), and Arg-Ser-Gly-Phe-Tyr (RSGFY, MW = 555.27) were prepared to examine the capability of 9AA/CDs matrix for the determination of short-chain peptides. As shown in Figure 4c, only two peptides were analyzed using 9AA as matrix, and four peptides were detectable but with a fairly low response when using 9AA/CHCA. However, all of the peptides were well detected in the form of [M + Na]+ when using 9AA/CDs as matrix. Interestingly, the [M + H]+ ion was also detected for RSGFY with high signal intensity. The phenomenon might be caused by the guanidine group (–CN3H4) of Arg that serves a as strong proton-accepter and is easily protonated during the ionization process. As we know, saccharide is one type of metabolite that plays a crucial role in physiological and pathologic processes. However, the analysis of saccharides by mass spectrometry was

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Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

Figure 4. MALDI-TOF mass spectra of (a) nucleosides (cytidine, m/z 266.218, [M + Na]+; uridine, m/z 283.223, [M + K]+; adenosine, m/z 290.231, [M + Na]+; guanosine, m/z 306.234, [M + Na]+, m/z 322.225, [M + K]+); (b) amino acids (Pro, m/z 138.086, [M + Na]+, m/z 154.102, [M + K]+; Asn, m/z 155.122, [M + Na]+, m/z 171.211, [M + K]+, m/z 176.987, [M + 2Na – H]+; Met, m/z 172.198, [M + Na]+; His, m/z 178.091, [M + Na]+; Glu, m/z 185.995, [M + Na]+); (c) peptides (YGG, m/z 318.224, [M + Na]+; YF, m/z 351.256, [M + Na]+; FGFG, m/z 449.254, [M + Na]+; RSGFY, m/z 629.242, [M + H]+, m/z 651.409, [M + Na]+ ), and (d) oligosaccharides (glucose, m/z 181.256, [M + H]+, m/z 219.224, [M + K]+; lactose, m/z 365.311, [M + Na]+, m/z 387.324, [M + 2Na – H]+; raffinose, m/z 527.419, [M + Na]+, m/z 543.427, [M + K]+) analyzed with 9AA (black Line), 9AA/CHCA (blue line) and 9AA/CDs (red line), respectively, in the low molecular weight region in positive ion mode. The signal peak at m/z 195 is [M + H]+ of 9AA matrix. The amount of each analyte is set as 500 pmol. Laser intensity: 60%

hindered due to their poor ionization efficiency [34]. Therefore, fast and convenient detection of saccharides is necessary. Herein, the oligosaccharides, including glucose (MW = 180.16), lactose (MW = 342.30), and raffinose (MW = 504.44) were chosen for MALDI MS analysis. In details, the binary matrix 9AA/CHCA exhibited a poorer analytical performance than those of 9AA; the probable reason was that the oligosaccharides could not be determined using CHCA matrix. Conversely, the signal intensities of [M + K]+ ions of glucose and [M + Na]+ ions of lactose and raffinose were greatly increased when using 9AA/CDs as matrix, and their S/N ratios increased about 2.5 times compared with those of 9AA, demonstrating the feasibility of this binary matrix for oligosaccharides analysis (Figure 4d). Moreover, 9AA/CDs matrix was then extended for the analysis of anticancer drugs, including doxorubicin, curcumin, paclitaxel, and etoposide, showing a wider range of LMW compounds application (Supplementary Figure S5). The above results demonstrated that 9AA/CDs is an efficient binary matrix that can be broadly applied for small molecules detection in positive ion MALDI MS.

Proposed Mechanism of Improved LDI Efficiency Based on the good performance of 9AA/CDs, the proposed mechanism of the binary matrix for improving the ionization efficacy was further explored. First, in the expanded XPS spectra of CDs shown in Supplementary Figure S2, the C1s peaks at 284.8 and 288.5 eV were identified as sp2 bonded carbon in the form of C=C and C=O respectively, validating the existence of the continuous π-conjugated structure of CDs. Meanwhile, the O1s peak at 531.9 eV and 533.2 eV might indicate that oxygen is mostly in the form of C=O and C-O, which demonstrated the abundant carboxyl and hydroxyl groups on the surface of CDs. As one major component of CDs dopants, oxygen atoms in the form of carboxyl and hydroxyl groups could act as a Lewis acid and tended to transfer protons to the analytes upon their desorption. This might help 9AA increase ionization degree of the analytes by MALDI-TOF MS in positive ion mode. In addition, the matrix must exhibit a strong absorption of photons at the laser emission wavelength (337 nm) to aid the ionization of analyte

COOH

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Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

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Figure 5. The proposed mechanism of improved MALDI efficiency. (a) UV-vis absorption spectra of 9AA, CDs, and 9AA/CDs solution. The inset shows expanded spectra from the range of 250–400 nm. (b) Schematic of the mechanism of 9AA/CDs as a matrix to improve the detection sensitivity of small molecules by positive ion MALDI-TOF MS

molecules in the gas phase. As shown in Figure 5a, 9AA/CDs solution exhibited a stronger absorbance near the wavelength of 337 nm than those of their two singular matrices, which was aiding better laser absorption and energy transfer from the UV laser beam. This phenomenon might be due to the continuous π-conjugated network and hydrogen-bond interaction between 9AA and CDs (Figure 5b). Moreover, CDs supported 9AA to form a thin and uniform layer of matrix crystal, which might help to achieve high sensitivity for the LDI analysis [35] (Figure 3c and d). Finally, CDs

could serve as a matrix additive used to suppress 9AA ionization and improve the S/N of analytes. For example, as shown in Figure 1d, [M + K]+ ions of Gln at m/z 185.134 could not be acquired because of the strong matrix suppression when using 9AA as matrix. However, adding CDs as a competitor for ionization drastically reduced the matrix background in the MS spectrum and the signal at m/z 185.134 with high S/N was clearly observed. The above synergistic effects might lead to better signal intensities of small molecules obtained by 9AA/CDs matrix.

Figure 6. MALDI-TOF MS mass spectrum of glucose and nucleosides in urine sample with 9AA/CDs as matrix. Glucose (m/z 181.056, [M + H]+), thymidine (m/z 243.178, [M + H]+, m/z 265.184, [M + Na]+), uridine (m/z 267.209, [M + H]+), cytidine (m/z 266.201, [M + Na]+, m/z 282.207, [M + K]+), acetylcytidine (m/z 286.155, [M + H]+), and guanosine (m/z 306.224, [M + Na]+) were identified. The inset presents the relationship between the ion intensity of the peak m/z 266.201 [M + Na]+ and the concentration of cytidine

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Determination of Glucose and Nucleosides in Urine Sample To further examine the applicability of 9AA/CDs binary matrix, cis-diols of a urine sample from a heathy female was extracted and followed by MALDI-TOF MS analysis. As shown in Figure 6, the molecular ion peaks identified in the mass spectra might be glucose (m/z 181.056, [M + H]+), thymidine (m/z 243.178, [M + H]+, m/z 265.184, [M + Na]+), uridine (m/z 267.209, [M + H]+), cytidine (m/z 266.201, [M + Na]+, m/z 282.207, [M + K]+), N4-acetylcytidine [36] (m/z 286.155, [M + H]+), and guanosine (m/z 306.224, [M + Na]+). In order to confirm their structures, the tandem mass spectra of the urine samples obtained from MALDI-TOF/TOF MS/MS were compared with those of their corresponding standards. The related information including the mass obtained from the mass spectra, the calculated mass, and fragment ions of the metabolites are summarized in Supplementary Table S2. Importantly, comparing the fragmentation patterns of the standards (except N4-acetylcytidine) with those from urine samples in the MS/MS spectra, all the five metabolites could be validated because more than 80% ion peaks were matched (Supplementary Figure S6). Then, the fragments of N4-acetylcytidine was identified manually by the ions of [M – CH3CO + H]+ (m/z 243.352) and [M – CH3CONH + H]+ (m/z 228.215). Moreover, cytidine was selected as a model to demonstrate the feasibility of the 9AA/CDs matrix for quantification. As shown in the inset of Figure 6, the calibration curve was plotted according to the intensity of [cytidine + Na]+ with regression equation of y = 2873x + 826. Good linearity was achieved with R2 > 0.99 in a wide concentration range of 0.5– 100 μM, and the concentration of cytidine was quantified with 0.66 μM (0.1605 μg mL–1) in the urine sample. This method not only simplified the quantification process but also made the result more reliable as the peaks produced by the internal standards often closely and easily affect the intensity of analytes. Therefore, the 9AA/CDs matrix has been demonstrated to be feasible for detection and quantification of LMW compounds in real sample.

Conclusion CDs was synthesized and employed as a novel binary matrix with 9AA for small molecules analysis by positive ion MALDI MS. A wide range of small molecules comprising nucleosides, amino acids, oligosaccharides, peptides, and some anticancer drugs were investigated. Compared with 9AA and 9AA/ CHCA matrices, the binary matrix 9AA/CDs exhibited several advantages, such as its relatively low matrix interferences, low fragmentation, high detection sensitivity, and wide scope for small molecules detection. Moreover, a more homogenous matrix-analyte co-crystal and better reproducibility were also demonstrated with the optimized solvent. The better analytical performance of 9AA/CDs was attributed to the π-conjugated network, the abundant carboxyl and hydroxyl groups on the

Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

CDs surface, the thin layer of matrix crystal, and 9AA ionization suppression by CDs. The good performance of this binary matrix provided the possibility for the determination of nucleosides, glucose, and the clinical monitoring of cytidine level in urine sample. In summary, it is believed that the 9AA/CDs could be an efficient and feasible matrix to solve the analytical challenges of reproducibility and sensitivity for MALDI-TOF MS analysis.

Acknowledgments This work is supported by the National Natural Science Foundation of China (Nos. 21305074 and 21475073), the Natural Science Foundation of Guangdong Province (No. 2014A030313757), and Shenzhen Municipal Government SZSITIC (Nos. CXB201104210014A and JCYJ20140902110354248).

References 1. Karas, M., Hillenkamp, F.: Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60, 2299–2301 (1988) 2. Deng, Z., Ye, M., Bian, Y., Liu, Z., Liu, F., Wang, C., Qin, H., Zou, H.: Multiplex isotope dimethyl labeling of substrate peptides for high throughput kinase activity assay via quantitative MALDI MS. Chem. Commun. 50, 13960–13962 (2014) 3. Velickovic, D., Herdier, H., Philippe, G., Marion, D., Rogniaux, H., Bakan, B.: Matrix-assisted laser desorption/ionization mass spectrometry imaging: a powerful tool for probing the molecular topology of plant cutin polymer. Plant J. 80, 926–935 (2014) 4. Vaidyanathan, S., Goodacre, R.: Quantitative detection of metabolites using matrix-assisted laser desorption/ionization mass spectrometry with 9-aminoacridine as the matrix. Rapid Commun. Mass Spectrom. 21, 2072– 2078 (2007) 5. Liu, H., Chen, R., Wang, J., Chen, S., Xiong, C., Wang, J., Hou, J., He, Q., Zhang, N., Nie, Z., Mao, L.: 1,5-Diaminonaphthalene hydrochloride assisted laser desorption/ionization mass spectrometry imaging of small molecules in tissues following focal cerebral ischemia. Anal. Chem. 86, 10114–10121 (2014) 6. Yang, H., Wang, J., Song, F., Zhou, Y., Liu, S.: Isoliquiritigenin (4,2',4'trihydroxychalcone): a new matrix-assisted laser desorption/ionization matrix with outstanding properties for the analysis of neutral oligosaccharides. Anal. Chim. Acta 701, 45–51 (2011) 7. Chen, R., Chen, S., Xiong, C., Ding, X., Wu, C.C., Chang, H.C., Xiong, S., Nie, Z.: N-(1-naphthyl) ethylenediamine dinitrate: a new matrix for negative ion MALDI-TOF MS analysis of small molecules. J. Am. Soc. Mass Spectrom. 23, 1454–1460 (2012) 8. Miura, D., Fujimura, Y., Tachibana, H., Wariishi, H.: Highly sensitive matrix-assisted laser desorption ionization-mass spectrometry for highthroughput metabolic profiling. Anal. Chem. 82, 498–504 (2010) 9. Wei, J., Buriak, J.M., Siuzdak, G.: Desorption-ionization mass spectrometry on porous silicon. Nature 399, 243–246 (1999) 10. McLean, J.A., Stumpo, K.A., Russell, D.H.: Size-selected (2–10 nm) gold nanoparticles for matrix assisted laser desorption ionization of peptides. J. Am. Chem. Soc. 127, 5304–5305 (2005) 11. Lin, Z., Zheng, J., Bian, W., Cai, Z.: CuFe2O4 magnetic nanocrystal clusters as a matrix for the analysis of small molecules by negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Analyst 140, 5287–5294 (2015) 12. Shrivas, K., Hayasaka, T., Sugiura, Y., Setou, M.: Method for simultaneous imaging of endogenous low molecular weight metabolites in mouse brain using TiO2 nanoparticles in nanoparticle-assisted laser desorption/ ionization-imaging mass spectrometry. Anal. Chem. 83, 7283–7289 (2011) 13. Yi, Z.L., Zhang, W., Xin, U.L., Bai, Y., Zhou, H.H., Liu, H.W.: A lithiumrich composite metal oxide used as a SALDI-MS matrix for the determination of small biomolecules. Chem. Commun. 50, 15397–15399 (2014)

Y. Chen et al.: 9AA/CDS as a Binary Matrix for MALDI MS

14. Xu, S., Li, Y., Zou, H., Qiu, J., Guo, Z., Guo, B.: Carbon nanotubes as assisted matrix for laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 75, 6191–6195 (2003) 15. Tang, H.W., Ng, K.M., Lu, W., Che, C.M.: Ion desorption efficiency and internal energy transfer in carbon-based surface-assisted laser desorption/ ionization mass spectrometry: desorption mechanism(s) and the design of SALDI substrates. Anal. Chem. 81, 4720–4729 (2009) 16. Coffinier, Y., Szunerits, S., Drobecq, H., Melnyk, O., Boukherroub, R.: Diamond nanowires for highly sensitive matrix-free mass spectrometry analysis of small molecules. Nanoscale 4, 231–238 (2012) 17. Sunner, J., Dratr, E., Chen, Y.-C.: Graphite surface-assisted laser desorption/ionization time-of-flight mass spectrometry of peptides and proteins from liquid solutions. Anal. Chem. 67, 4335–4342 (1995) 18. Meng, J., Shi, C., Deng, C.: Facile synthesis of water-soluble multi-wall carbon nanotubes and polyaniline composites and their application in detection of small metabolites by matrix assisted laser desorption/ ionization mass spectrometry. Chem. Commun. 47, 11017–11019 (2011) 19. Dong, X., Cheng, J., Li, J., Wang, Y.: Graphene as a novel matrix for the analysis of small molecules by MALDI-TOF MS. Anal. Chem. 82, 6208– 6214 (2010) 20. Szabo, Z., Vallant, R.M., Takátsy, A., Bakry, R., Najam-ul-Haq, M., Rainer, M., Huck, C.W., Bonn, G.K.: Laser desorption/ionization mass spectrometric analysis of small molecules using fullerene-derivatized silica as energy-absorbing material. J. Mass Spectrom. 45, 545–552 (2010) 21. Xu, X., Ray, R., Gu, Y., Ploehn, H.J., Gearheart, L., Raker, K., Scrivens, W.A.: Electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments. J. Am. Chem. Soc. 126, 12736– 12737 (2004) 22. Miao, P., Han, K., Tang, Y., Wang, B., Lin, T., Cheng, W.: Recent advances in carbon nanodots: synthesis, properties and biomedical applications. Nanoscale 7, 1586–1595 (2015) 23. Wei, W., Xu, C., Wu, L., Wang, J., Ren, J., Qu, X.: Nonenzymatic browning reaction: a versatile route for production of nitrogen-doped carbon dots with tunable multicolor luminescent display. Sci. Rep. 4, 3564 (2014) 24. Chen, S., Zheng, H., Wang, J., Hou, J., He, Q., Liu, H., Xiong, C., Kong, X., Nie, Z.: Carbon nanodots as a matrix for the analysis of low-molecular-weight molecules in both positive- and negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantification of glucose and uric acid in real samples. Anal. Chem. 85, 6646–6652 (2013)

1235

25. Guo, Z., He, L.: A binary matrix for background suppression in MALDIMS of small molecules. Anal. Bioanal. Chem. 387, 1939–1944 (2007) 26. Zhou, L.-H., Kang, G.-Y., Kim, K.P.: A binary matrix for improved detection of phosphopeptides in matrix-assisted laser desorption/ ionization mass spectrometry. Rapid Commun. Mass Spectrom. 23, 2264–2272 (2009) 27. Shanta, S.R., Zhou, L.H., Park, Y.S., Kim, Y.H., Kim, Y., Kim, K.P.: Binary matrix for MALDI imaging mass spectrometry of phospholipids in both ion modes. Anal. Chem. 83, 1252–1259 (2011) 28. Calvano, C.D., Monopoli, A., Ditaranto, N., Palmisano, F.: 1,8-bis(dimethylamino)naphthalene/9-aminoacridine: a new binary matrix for lipid fingerprinting of intact bacteria by matrix assisted laser desorption ionization mass spectrometry. Anal. Chim. Acta 798, 56–63 (2013) 29. Baker, S.N., Baker, G.A.: Luminescent carbon nanodots: emergent nanolights. Angew. Chem. Int. Ed. Engl. 49, 6726–6744 (2010) 30. Guo, Y., Wang, Z., Shao, H., Jiang, X.: Hydrothermal synthesis of highly fluorescent carbon nanoparticles from sodium citrate and their use for the detection of mercury ions. Carbon 52, 583–589 (2013) 31. Dong, Y., Pang, H., Yang, H.B., Guo, C., Shao, J., Chi, Y., Li, C.M., Yu, T.: Carbon-based dots co-doped with nitrogen and sulfur for high quantum yield and excitation-independent emission. Angew. Chem. Int. Ed. Engl. 52, 7800–7804 (2013) 32. Zhao, A., Chen, Z., Zhao, C., Gao, N., Ren, J., Qu, X.: Recent advances in bioapplications of C-dots. Carbon 85, 309–327 (2015) 33. Wei, Y., Li, S., Wang, J., Shu, C., Liu, J., Xiong, S., Zhang, J., Zhao, Z.: Polystyrene spheres-assisted matrix-assisted laser desorption ionization mass spectrometry for quantitative analysis of plasma lysophosphatidylcholines. Anal. Chem. 85, 4729–4734 (2013) 34. Harvey, D.J.: Analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: an update for 2009– 2010. Mass Spectrom. Rev. 34(3), 268–422 (2014) 35. Gorka, J., Bahr, U., Karas, M.: Graphite supported preparation (GSP) of alpha-cyano-4-hydroxycinnamic acid (CHCA) for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for peptides and proteins. J. Am. Soc. Mass Spectrom. 23, 1949–1954 (2012) 36. Willmann, L., Erbes, T., Krieger, S., Trafkowski, J., Rodamer, M., Kammerer, B.: Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism. Anal. Bioanal. Chem. 407, 3555–3566 (2015)