Quantum Dot Enhancement of Peptide Detection by Matrix-Assisted

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Quantum Dot Enhancement of Peptide Detection by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Chih-Wei Liu,† Min-Wei Chien,† Guo-Feng Chen,§ Shun-Yuan Chen,†,‡ Chih-Sheng Yu,‡ Ming-Yuan Liao,*,§ and Chien-Chen Lai*,†,^ †

Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan Instrument Technology Research Center, National Applied Research Laboratories, Taiwan § Department of Chemistry, National Chung Hsing University, Taichung, Taiwan ^ Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan ‡

bS Supporting Information ABSTRACT: Matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOFMS) is a rapid and sensitive tool for characterizing a wide variety of biomolecules. However, invisible “sweet spots” that form during heterogeneous cocrystallization minimize the analytical throughput and affect the reproducibility of MALDI analysis. In this study, visible “sweet spots” were generated on a metallic sample plate by quantum dots (QDs)-assisted MALDI analysis. To the best of our knowledge, this is the first report to demonstrate that “sweet spots” can be observed directly without using a microscope. The proper proportion of matrix to QDs that results in optimal crystallization was also examined. The signals of standard peptides and phosphopeptides obtained by QD-assisted MALDI analysis were 5- and 3-fold higher, respectively, than those obtained by conventional MALDI analysis. For peptide mixtures, the QD-assisted MALDI analysis not only resulted in more intense peptide signals but also resulted in a greater number of peaks, thereby allowing for better detection of individual peptides in peptide mixtures. Moreover, we demonstrated that application of QDs to a radiate microstructure chip followed by MALDI analysis enhanced the detection of peptide signals. Overall, we show that this method is a simple, sensitive, and high-throughput technique for peptide detection.

M

atrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is an important analytical tool for characterization of biomolecules.1 8 In MALDI, conventional organic matrixes (e.g., R-cyano-4-hydroxycinnamic acid, CHCA) absorb the energy of the laser pulse and then transfer it to the surrounding analytes for desorption and ionization. However, the heterogeneous cocrystallization of analytes and organic matrixes often leads to the formation of invisible “sweet spots” that affect the shot-to-shot reproducibility during MALDI analysis. At the same time, detection of “sweet spots” is a time-consuming process. Another limitation of MALDI is organic matrix interference in the low-mass region (m/z of 18 MΩ). Synthesis of Quantum Dots. L-Cysteine (1.9384 g) and Tris (7.7528 g) were dissolved in 50 mL of water in a wolff bottle, then 10 mL of zinc acetate (0.8 M) and 10 mL of manganese acetate (0.016 M) were added drop by drop to the above solution. Oxygen was then purged from the solution by N2 bubbling for 30 min, after which the L-cysteine Zn solution was reacted slowly with Na2S (20 mL, 0.8 M) at 98 C for 24 h to form Mn-ZnS@Cys QDs. Finally, the QDs were precipitated with ethanol (300 mL), collected by centrifugation, and then dried in a concentrator (miVac, Genevac, Ipswich, Suffolk, UK). The QDs were dissolved in water before being used. Powder X-ray diffraction (XRD) experiments were conducted to estimate the size of QDs. The experiments were carried out in an XRD-7000 diffractometer (Shimadzu). Sample Preparation. Each analytic sample was dissolved in QD buffer, and the control samples were dissolved in ddH2O. CHCA matrixes were dissolved in 50% ACN with 0.1% TFA. Samples and matrixes were prepared as reported elsewhere.27 Briefly, 1 μL of sample (with or without QDs) was mixed with an

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Table 1. Effect of Concentration of Mn-ZnS@Cys QDs for Peptide Detection on Metallic Sample Plate Using MALDI-MS Ang I (400 fmol/μL)

a

CHCA (mg/mL)

QDs (mg/mL)

intensitya

RSD (%)

10

10

15172.6

4.0

10

5

19422.3

5.2

10 10

2 1

21096.3 8806.0

5.5 6.7

10

0

4948.3

9.6

Average intensity (n = 3).

equal volume of CHCA, then 1 μL of analyte solution was deposited onto a stainless steel sample plate or the microstructure chip, air-dried, and analyzed in a Voyager-DE Pro MALDITOF mass spectrometer equipped with a pulsed nitrogen laser set at 337 nm (Applied Biosystems, Foster City, CA). Proteolytic In-Solution Digestion. Intact BSA protein (2 μg) was dissolved in 50 mM ammonium bicarbonate (ABC). The BSA solution was sequentially reduced with 10 mM DTT at 56 C for 1 h and was alkylated with 55 mM IAA at 37 C in the dark for 30 min. Both DTT and IAA were dissolved in 50 mM ABC. Proteolytic digestion was carried out by incubating 40 ng of trypsin (Trypsin Gold, Promega, Madison, WI, USA) in 50 mM ABC for 16 h at 37 C. The tryptic peptide solution was dried in a concentrator (miVac, Genevac, UK) and was used for MS analysis without further purification. MS Analysis. All mass spectra were acquired in the linear positive or negative ion mode with delayed extraction. The extraction and guide wire voltages were set at 20 kV and 0.05%. Five profiles containing 200 shots each from the same spot were acquired. The mass calibration was achieved using ion peaks of Ang I, T6, and BSA digests. Finally, data analysis was performed by Data Explorer software (Applied Biosystems).

’ RESULTS AND DISCUSSION Characterization of Mn-ZnS@Cys QDs. XRD is a wellknown technique that is used to characterize the lattice properties, particle size, and structure of QDs. The XRD patterns of the QDs in this study are shown in Figure S1-A in the Supporting Information. Three characteristic peaks (namely, 111, 220, and 311) correlated with the cubic structure of Mn2+-doped ZnS. From this diffractogram, the particle size of the QDs was estimated to be 5.1 nm by the Scherrer equation.28 Figure S1-B in the Supporting Information shows the excitation and emission spectra of QDs (λex = 320 nm). The peak at 590 nm confirmed the existence of QDs. Figure S1-C in the Supporting Information shows the UV vis spectra of QDs. The high absorption wavelength was 290 nm, which is close to 337 nm, indicating that these QDs could possibly serve as an effective matrix for MALDI analysis. QD-Assisted MALDI Analysis of Peptides and Phosphopeptides. At first, we examined whether QDs could serve as a good matrix for peptide analysis. Results of the SALDI analysis revealed that a high concentration of Ang I (10 pmol/μL) could not be detected when the QDs (2 mg/mL) alone served as the matrix (data not shown). We then tested whether QDs combined with CHCA as the matrix (QD-assisted MALDI analysis) could improve the sensitivity of peptide detection. As shown in 6594

dx.doi.org/10.1021/ac201016c |Anal. Chem. 2011, 83, 6593–6600

Analytical Chemistry

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Table 2. Effect of Mn-ZnS@Cys QDs for Phosphopeptide T6 Detection on Metallic Sample Plate and Microstructure Chip Using MALDI-MS T6 (2 pmol/μL) CHCA

QDs

(mg/mL)

(mg/mL)

metallic sample plate microstructure chip a

Figure 1. MALDI mass peak intensity spectra obtained from Ang I (A, B) and T6 (C, D) on the metallic sample plate. The matrixes included CHCA (A, C), and CHCA combined with QDs (B, D), respectively.

Figure 1A,B, Ang I (400 fmol/μL) signals were significantly stronger when the matrix for MALDI analysis comprised QDs and CHCA than when the matrix comprised CHCA alone. We also evaluated the effect of concentration of QDs (1, 2, 5, and 10 mg/mL) on peptide detection and found that 2 mg/mL was the optimal concentration (Table 1, Figure S-2 in the Supporting Information). At that concentration, Ang I signals obtained by QD-assisted MALDI analysis were 5-fold higher than those obtained when the matrix comprised only CHCA. We therefore chose 2 mg/mL QDs for the following experiments. The monophosphopeptide T6 (2 pmol/μL) was also used to test whether QD-assisted MALDI analysis could improve the signal intensities of phosphopeptides (Figure 1C,D). We found that the signal intensities of T6 obtained by QD-assisted MALDI analysis

intensitya

RSD (%)

10

2

16013.3

14.5

10

0

5156.7

9.3

2

0.05

31756.7

3.2

2

0

14542.0

4.2


Figure 2. MALDI mass spectra obtained from BSA digests. The matrixes included CHCA only (A) and CHCA combined with QDs (B). The open circles indicate the peaks that were detected only in QDassisted MALDI analysis. The open and solid stars indicate less than or more than a 1.5-fold improvement in signals, respectively.

were 3-fold higher than those obtained from MALDI analysis when the matrix comprised CHCA alone (Table 2). The above-mentioned data imply that a matrix composed of QDs and CHCA improves the sensitivity of detection of peptides and phosphopeptides. Analysis of Peptide Mixtures. We then assessed the feasibility of QD-assisted MALDI analysis of peptide mixtures. Standard BSA digests (1 pmol/μL) were analyzed by QD-assisted MALDI analysis (2 mg/mL) or by MALDI comprising CHCA alone. The resulting MALDI spectra were then subjected to a MASCOT search (http://www.matrixscience.com/). The matching peaks are listed in Table S1 in the Supporting Information. MALDI comprising CHCA alone resulted in 16 peaks (Figure 2A), and QD-assisted MALDI analysis resulted in a total of 19 peaks (Figure 2B). QD-assisted MALDI analysis of samples on the 6595



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Figure 3. MALDI mass spectra of T6 mixed with BSA digests analyzed in the positive (A F) and negative (G L) mode. The matrixes included CHCA only (A, C, E, G, I, and K), and CHCA combined with QDs (B, D, F, H, J, and L).

metallic sample plate resulted in three unique peaks (m/z 1462.6, 1510.8, and 1531.8). In addition, the signals of 12 of the 16 peaks that were common to both spectra were 1.5 4.2 times more intense on the QD-assisted MALDI spectrum than on the spectrum generated by MALDI comprising CHCA alone. Therefore, QD-assisted MALDI analysis not only results in more intense peptide signals but also results in a greater number of peaks. Those data indicate that QD-assisted MALDI analysis is excellent at detecting individual peptides in peptide mixtures. In addition, QD-assisted MALDI analysis of samples on our radiate

microstructure chip showed that the signals from samples on our chip were significantly greater than those on the unmodified plate. Next, we evaluated whether QD-assisted MALDI analysis can increase the intensity of phosphopeptide signals for “bottom-up” proteomics. BSA digests (1 pmol/μL) were mixed with a series of concentrations of T6 (1, 0.5, and 0.2 pmol/μL) to mimic phosphoprotein digests. At all concentrations of T6, QD-assisted MALDI analysis showed higher signal intensities, even in the presence of BSA peptides (Figure 3 and Table S2 in the Sup6596



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Figure 5. Relationship between the signal intensity and the cocrystalline morphology in the QD-assisted MALDI analysis. (A) The formation of cocrystalline structures in QD-assisted MALDI analysis was observed under a video monitor. The dark center zone was the location of “sweet spots”. (B) The signals were obtained from the locations along the horizontal red line. Each data point was accumulated with 150 laser shots.

Figure 4. Micrographs showing cocrystalline morphology of CHCA + Ang I (A), CHCA + QDs (B), and CHCA + QDs + Ang I (C). The dark arrow indicates the locations of sweet spots formed by QDs.

porting Information). Similar data were obtained in both positive and negative detection modes, indicating that QD-assisted MALDI analysis has the remarkable ability to enhance the signal intensities of phosphopeptides within peptide mixtures. The QDassisted MALDI signals of the T6 phosphopeptide were markedly more intense in the negative detection mode. Overall, increased signal intensities and improved S/N ratios of T6 in BSA digests were obtained using QD-assisted MALDI analysis. Furthermore, consistent results were obtained at even lower concentrations of T6. The data strongly indicate that QD-assisted MALDI analysis results in enhanced phosphopeptide signals, even in the presence of BSA digests. Cocrystalline Morphology: How Did QDs Affect the Signals of Analytes? During the MALDI analysis, we used a video monitor to observe the formation of cocrystalline structures of CHCA

and QDs mixtures. Figure 4B C shows that the “sweet spots” on the metallic sample plate were visible through an optical microscope. Signals emanating from those spots were 10 times more intense than those emanating from other regions (Figue 5). Pulsed nitrogen laser shot at the locations along the horizontal red-line labeled in Figue 5, and the intense signals were significantly obtained from the locations of the visible “sweet spots”. In this study, the standard peptides were dissolved directly in the QD buffer and then mixed with CHCA. It is possible that the QDs can absorb or trap the peptides at first through the interactions between analytes and the cysteine capping agents on the surfaces of QDs, thereby increasing the relative concentrations of peptides. It is also possible that Mn2+-doped ZnS QDs act as nucleation centers for the aggregation of visible sweet spots during matrix crystallization. We found that the intensity of the signals emanating from the visible sweet spots was significantly greater than the intensity of signals emanating from the conventional CHCA matrix. To the best of our knowledge, this is the first report to show that sweet spots can be formed by QDs and that they can be observed directly using a video monitor. Moreover, QD-assisted MALDI analysis does not require time-dependent enrichment steps. On the basis of our results, we conclude that QD-assisted MALDI analysis is a simple but effective technique for detecting peptides. QD-Assisted MALDI Analysis of Tryptic BSA Digests from Proteolytic in-Solution Digestion. We then assessed the feasibility of QD-assisted MALDI analysis of tryptic BSA digests. Tryptic peptides were generated from intact BSA protein as 6597



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Table 3. Effect of Concentration of Mn-ZnS@Cys QDs for Peptide Detection on Microstructure Chip Using MALDI-MS Ang I (400 fmol/μL) CHCA (mg/mL)

a

QDs (mg/mL)

intensity

RSD (%)

2

2

4135.2

15.6

2

0.5

10853.0

18.4

2 2

0.1 0.05

18816.3 46596.3

4.9 9.0

2

0.01

13775.3

9.8

2

0

23690.3

7.4


described in the Materials and Methods. The tryptic peptides (0.8 pmol/μL) were analyzed by QD-assisted MALDI analysis (2 mg/mL) or by MALDI comprising CHCA alone (Figure S3 in the Supporting Information). QD-assisted MALDI analysis resulted not only in more intense peptide signals but also in a greater number of peaks. Those data demonstrate that QDassisted MALDI analysis is an effective technique for detecting peptides. Moreover, in our experience, less than 30 s is needed to obtain MALDI spectra by QD-assisted MALDI analysis through focusing on visible “sweet spots”. QD-assisted MALDI analysis is, therefore, a rapid and excellent technique for the detection of peptides and phosphopeptides. QD-Assisted MALDI Analysis of Samples Concentrated on a Radiate Microstructure MALDI Chip. We previously developed a radiate microstructure MALDI chip on which analytes can be concentrated directly, resulting in improved detection of peptide signals.27 In this study, we applied QD-assisted MALDI analysis of analytes on the microstructure MALDI chip. As shown in Figure S-4A B in the Supporting Information, signals of Ang I (400 fmol/μL) were significantly more intense on the microstructure chip when analyzed by QD-assisted MALDI analysis (QDs, 0.05 mg/mL) than when analyzed using MALDI comprising CHCA alone (CHCA, 2 mg/mL). The results showed that QD-assisted MALDI analysis was further improved by our radiate microstructure MALDI chip (Tables 1 3 and Figure S4B in the Supporting Information). QD-assisted MALDI analysis (Table 3 and Figure S-3B in the Supporting Information). We also evaluated the effect of the concentration of the QDs (0.01, 0.05, 0.1, 0.5, and 2 mg/mL) for peptide detection and crystallization and found that 0.05 mg/mL QDs was the optimal concentration for the microstructure chip (Table 3 and Figure S5 in the Supporting Information). The signals for Ang I obtained by QD-assisted MALDI analysis were 2-fold higher than those obtained by MALDI analysis comprising CHCA only. Therefore, we chose 0.05 mg/mL as the QD concentration in the following experiments. The scanning electron micrographs of the visible “sweet spots” on the QD-assisted MALDI microstructure chip are shown in Figure 6B. T6 (2 pmol/μL) was also used to test whether QD-assisted MALDI would result in higher signal intensities of phosphopeptides on the microstructure chip (Figure S-4C D in the Supporting Information). We observed that the T6 signals detected by QD-assisted MALDI analysis were 2 times more intense than those detected by MALDI comprising CHCA only (Table 2). The results imply that a matrix comprising

Figure 6. SEM images of CHCA only (A) and CHCA + QDs + Ang I (B). The dark arrow indicates the locations of sweet spots formed by QDs.

QDs and CHCA improves the signals emitted by peptides and phosphopeptides. We also assessed whether QD-assisted MALDI analysis could enhance the signal strength of various peptides in peptide mixtures on the microstructure chip. BSA digests (1 pmol/μL) were analyzed by QD-assisted MALDI analysis (0.05 mg/mL), and the MALDI spectra of BSA digests were subjected to a MASCOT search. The matching peaks are listed in Table S1 in the Supporting Information. MALDI comprising CHCA alone resulted in 15 peaks (Figure 7A), and QD-assisted MALDI analysis resulted in 24 peaks (Figure 7B). We found that 10 of the peaks generated by QD-assisted MALDI analysis were 1.5 times more intense than those generated by MALDI comprising CHCA alone. Overall, QD-assisted MALDI analysis on the microstructure chip resulted in more intense peptide signals. Moreover, we also evaluated whether QD-assisted MALDI analysis could enhance the phosphopeptide signals during “bottom-up” proteomics. BSA digests (1 pmol/μL) were mixed with a series of concentrations (1, 0.5, and 0.2 pmol/μL) of T6 to mimic phosphoprotein digests. We found that QD-assisted MALDI analysis of the mixtures on the microstructure chip yielded a high-intensity signal representing T6 (Figure S6 and Table S3 in the Supporting Information). Similar data were acquired in both positive and negative detection modes, although the intensity of the phosphopeptide signals was greater in the negative detection mode. These findings indicate that QD-assisted MALDI analysis has the remarkable 6598



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Figure 7. MALDI mass spectra obtained from BSA digests on the microstructure chip. The matrixes included CHCA only (A) and CHCA combined with QDs (B). The open circle indicates the peaks that existed only in QD-assisted MALDI analysis. The open and solid stars indicate less than or more than a 1.5-fold improvement in signals, respectively.

ability to enhance the signal intensities of phosphopeptides within peptide mixtures.

’ CONCLUSIONS In this study, we present a simple, sensitive, and highthroughput method for detecting peptides. Visible “sweet spots” on analyte/matrix crystals can be generated through cocrystallization during Mn2+-doped ZnS@Cys QD-assisted MALDI analysis and can be observed directly using a video monitor. On the metallic sample plate, the peptide signals and the phosphopeptide signals obtained by QD-assisted MALDI analysis were 5- and 3-fold higher than those obtained by conventional MALDI analysis, respectively. In addition, more intense signals and a greater number of peaks in the BSA digest spectra were acquired by QD-assisted MALDI analysis. On the radiate microstructure MALDI chip, the signals of peptides and phosphopeptides obtained by QD-assisted MALDI analysis were 2-fold higher than those obtained by conventional MALDI analysis. Finally, QD-assisted MALDI analysis of

peptide mixtures yields significantly elevated peptide and phosphopeptide signals.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*(C.-C.L.) Address: Institute of Molecular Biology, National Chung Hsing University, No. 250, Kuo-Kuang Road, Taichung, 402 Taiwan. Tel: (886) 4-22840485, ext 235. Fax: (886) 4-22858163. E-mail: [email protected]. (M.-Y.L.) Address: Department of Chemistry, National Chung Hsing University, No. 250, KuoKuang Road, Taichung, 402 Taiwan. Phone: (886) 4-22840411, ext 626. Fax: (886) 4-22862547. E-mail: [email protected]. edu.tw. 6599



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’ ACKNOWLEDGMENT The study was funded by a grant from the National Research Council of the Republic of China. C.-W.L. and M.-W.C. contributed equally to this work. ’ REFERENCES (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. (2) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (3) Egelhofer, V.; B€ussow, K.; Luebbert, C.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 2741–2750. (4) Hostetter, A. A.; Chapman, E. G.; DeRose, V. J. J. Am. Chem. Soc. 2009, 131, 9250–9257. (5) Finke, B.; Stahl, B.; Pfenninger, A.; Karas, M.; Daniel, H.; Sawatzki, G. Anal. Chem. 1999, 71, 3755–3762. (6) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349–450. (7) Egelhofer, V.; Gobom, J.; Seitz, H.; Giavalisco, P.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2002, 74, 1760–1771. (8) Lay, J. O. Mass Spectrom. Rev. 2001, 20, 172–194. (9) Sunner, J.; Dratz, E.; Chen, Y. C. Anal. Chem. 1995, 67, 4335–4342. (10) Amini, N.; Shariatgorji, M.; Thorsen, G. J. Am. Soc. Mass Spectrom. 2009, 20, 1207–1213. (11) Wada, Y.; Yanagishita, T.; Masuda, H. Anal. Chem. 2007, 79, 9122–9127. (12) Hsu, W. Y.; Lin, W. D.; Hwu, W. L.; Lai, C. C.; Tsai, F. J. Anal. Chem. 2010, 82, 6814–6820. (13) Kailasa, S. K.; Kiran, K.; Wu, H. F. Anal. Chem. 2008, 80, 9681–9688. (14) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912–5919. (15) Rodthongkum, N,; Chen, Y,; Thayumanavan, S,; Vachet, R. W. Anal. Chem. 2010, 82, 3686–3691. (16) Castellana, E. T.; Russell, D. H. Nano Lett. 2007, 7, 3023–3025. (17) Chiu, T. C.; Chang, L. C.; Chiang, C. K.; Chang, H. T. J. Am. Soc. Mass Spectrom. 2008, 19, 1343–1346. (18) Sherrod, S. D.; Diaz, A. J.; Russell, W. K.; Cremer, P. S.; Russell, D. H. Anal. Chem. 2008, 80, 6796–6799. (19) Chiang, C. K.; Yang, Z.; Lin, Y. W.; Chen, W. T.; Liu, H. J.; Chang, H. T. Anal. Chem. 2010, 82, 4543–4550. (20) Lorkiewicz, P.; Yappert, M. C. Anal. Chem. 2009, 81, 6596–6603. (21) Chiang, C. K.; Chiang, N. C.; Lin, Z. H.; Lan, G. Y.; Lin, Y. W.; Chang, H. T. J. Am. Soc. Mass Spectrom. 2010, 21, 1204–1207. (22) Shastri, L. A.; Kailasa, S. K.; Wu, H. F. Rapid Commun. Mass Spectrom. 2009, 23, 2247–2252. (23) Ke, Y.; Kailasa, S. K.; Wu, H. F.; Chen, Z. Y. Talanta 2010, 83, 178–184. (24) Shrivas, K.; Kailasa, S. K.; Wu, H. F. Proteomics 2009, 9, 2656–2667. (25) Bailes, J.; Vidal, L.; Ivanov, D. A.; Soloviev, M. J. Nanobiotechnology 2009, 7, 10. (26) Kailasa, S. K.; Wu, H. F. Analyst 2010, 135, 1115–1123. (27) Chen, S. Y.; Li, K. I.; Yu, C. S.; Wang, J. S.; Hu, Y. C.; Lai, C. C. Anal. Chem. 2010, 82, 5951–5957. (28) Cullity, B. D. Elements of X-ray Diffraction; Addison-Wesley: Boston, 1956, p 99. (29) Bhargava, R. N.; Gallagher, D.; Hong, X.; Nurmikko, A. Phy. Rev. Lett. 1994, 72, 416–419.

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Quantum Dot Enhancement of Peptide Detection by Matrix-Assisted

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