Proteins

Letter pubs.acs.org/ac

On-Plate Selective Enrichment and Self-Desalting of Peptides/ Proteins for Direct MALDI MS Analysis Zhoufang Zeng,† Yandong Wang,‡ Shoulei Shi,‡ Lifeng Wang,† Xinhua Guo,*,† and Nan Lu‡ †

College of Chemistry, Jilin University, Changchun 130012, China State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, China

‡

S Supporting Information *

ABSTRACT: In this paper, a new technique has been proposed to achieve simultaneous peptides/proteins enrichment and washfree self-desalting on a novel sample support with a circle hydrophobic−hydrophilic−hydrophobic pattern. Upon deposition, the sample solution is first concentrated in a small area by repulsion of the hydrophobic outer layer, and then, the peptides/ proteins and coexisting salt contaminants are selectively captured in different regions of the pattern through strong hydrophobic and hydrophilic attractions, respectively. As a result, the detection sensitivity is improved by 2 orders of magnitude better than the use of the traditional MALDI plate, and high-quality mass spectra are obtained even in the presence of NaCl (1 M), NH4HCO3 (100 mM), or urea (1 M). The practical application of this method is further demonstrated by the successful analysis of myoglobin digests with high sequence coverage, demonstrating the great potential in proteomic research.

M

hydrophobic spot, in which parts of the analytes are also diffused out of the hydrophobic spot at the same time.26 In this process, only small amount of samples can be applied on the spot, which may restrict the application of the method for the detection of trace peptides/proteins. Herein, a novel method for simultaneous peptides/proteins enrichment and wash-free self-desalting is demonstrated, in which a silicon wafer modified with a circle hydrophobic− hydrophilic−hydrophobic pattern is used as the sample support. Upon the sample application, the outermost hydrophobic layer prevents the sample from diffusion and focuses the samples in the small spot; additionally, the peptides/proteins and the salts are distributed in the central small hydrophobic space and the hydrophilic region, respectively. As a result, the detection sensitivity obtained with the use of the novel sample support is 2 orders of magnitude lower than that from the traditional MALDI plate. At the same time, high-quality mass spectra can be obtained even in the presence of NaCl (1 M), NH4HCO3 (100 mM), or urea (1 M). A main component of MS-based proteomics is protein identification in various biological samples, which relies on the mass spectra of digested peptides and the interpretation of the MS data using database-supported search engines.3 However, the complete detection of low-abundance digestion products is a challenge due to the interference from the introduced contaminants such as salts or other reagents.10 This leads to low sequence coverage that sometimes makes protein

atrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for the analysis of peptides/proteins that are present in various biological samples.1−4 Poor analyte−matrix cocrystallization influenced by uneven distribution of the analytes and low concentration of the analyte is the main problem for successful MALDI MS analysis.5,6 In addition, relatively high concentration of salts and other reagents coexisting in solution for maintaining structure and biological activities of the protein often result in a significant decrease of signal sensitivity and reproducibility.7−11 Therefore, an appropriate approach is critically required to achieve sample concentration and desalting before MALDI-MS analysis. Numbers of off-target methods including HPLC,12 ZiptipC18,13 and surface-functionalized nanoparticles10,14−18 have been used for prefractionation of complex biological samples before MALDI MS analysis. During the multiple sample handling steps, however, inevitable sample loss and potential contaminants are associated with the methods.6,8,19,20 Recently, hydrophobic polymers, such as Teflon,21 selfassembly monolayer (SAM),22 parafilm wax,23 polypropylene,24 and nylon25 have been developed as sample support for onplate peptides/proteins enrichment and desalting. However, an additional washing step is required, which causes sample loss and prevents the methods from being adapted for a highthroughput analysis.26 Yang et al. subsequently reported a rapid on-plate desalting and enrichment method, in which they templated the hydrophobic polymer in a small spot on the center of the MALDI plate.27 After the application of the sample onto the spot, an excessive volume of matrix solution is need to make the salts redissolve and flow out of the © 2012 American Chemical Society

Received: December 19, 2011 Accepted: February 10, 2012 Published: February 10, 2012 2118

dx.doi.org/10.1021/ac203378q | Anal. Chem. 2012, 84, 2118−2123

Analytical Chemistry

Letter

Scheme 1. Schematic Flowchart of Peptides/Proteins Enrichment and Self-Desalting Process on the Novel Sample Support

into the clean vial after aspirating and dispensing for several times. Fabrication of the Novel Sample Support. The silicon substrate was first cleaned in H2SO4/H2O2 (v/v = 3:1) at 80 °C for 30 min (caution: be careful). Second, a PMMA spot with size of about 800 μm in diameter was dip-coated on the cleaned silicon substrate, and then, the modified silicon surface was fluorinated with fluoroalkylsilane (heptadecafluoro-1,1,2,2tetrahydrodecyl trithoxysilane), followed by ultrasonication in acetone, ethanol, and water for 5 min in sequence to remove PMMA, which left an annular hydrophilic blank silicon circle with 800 μm in diameter. Finally, a smaller size PMMA spot (about 500 μm diameter) was dip-coated at the center of the circle. Mass Spectrometry Analysis. The fabricated sample support was attached onto the MALDI plate by double-side tap before sample deposition. One microliter of the sample solution was deposited onto the traditional MALDI plate or the novel sample support. After drying in air, 1 μL of matrix solution (10 mg mL−1 SA or DHB in a solution containing 50% (v/v) ACN and 0.1% (v/v) TFA) was added onto the dried analyte spot for MALDI analysis. MALDI-TOF MS experiment was performed in positive ion mode on a Kratos Axima CFRplus spectrometer (Shimadzu Biotech, Manchester, UK) with a 337 nm nitrogen laser and the acceleration voltage of 20 kV. Each spectrum was obtained by 150 laser shots. Search Parameters. The search parameters were as follows: database, SwissProt; digest used, Trypsin; maximum of missed cleavages: 1; peptide tolerance: ± 2 Da. Mascot from Matrix Science Ltd. (London, U.K.) was used to search all of the mass spectra.

identification difficult. The practical application of this method is further demonstrated by the successful analysis of peptides in myoglobin digests with high sequence coverage.

■

EXPERIMENTAL SECTION Chemicals and Materials. Bradykinin 1-7 fragment (Mw = 756.85), angiotensin II human (Mw = 1046.18), angiotensin III human (Mw = 931.10), lysozyme (Mw = 14400), myoglobin (Mw = 17000), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB), ZipTipC18, heptadecafluoro-1,1,2,2-tetrahydrodecyl trithoxysilane, poly(methyl methacrylate) (PMMA, Mw = 95000), and trypsin TPCK treated from bovine pancreas were purchased from Sigma-Aldrich. The silicon wafers (n type (100)) were obtained from Youyan Guigu (Beijing, China). The chemicals were of analytical grade. All aqueous solutions were prepared using Milli-Q water by Milli-Q system. Characterization. Scanning electron microscope (SEM) images were taken using environmental scanning electron microscope with an energy dispersive X-ray (EDX) characterization (ESEM, Model XL 30 ESEM FEG from Micro FEI Philips). The samples were sputtered with a thin layer of Pt prior to imaging (2 nm in thickness). Sample Preparation. Lysozyme and a mixture of three peptides including bradykinin 1-7 fragment, angiotensin II human, and angiotensin III human were dissolved in Milli-Q water to reach the concentration of 100 μM, respectively. The stock solution was further diluted for MALDI-TOF MS analysis. Protein Digestion. Myoglobin was dissolved in 100 mM ammonium bicarbonate (NH4HCO3) buffer at pH 8.0 and treated with trypsin (25:1, w/w) for 48 h at 37 °C. One microliter of formic acid was added into the solution to stop the reaction. Sample Preparation of ZipTipC18. Off-target desalting for peptide mixture was performed using ZipTipC18 pipet tips. In brief, ZipTipC18 was prewetted with 100% ACN by aspirate/ dispense step three times, and then, the tip was equilibrated by washing it twice with 0.1% TFA in Milli-Q water. Five microliters of sample solution was aspirated into the preequilibrated tip, followed by washing with 5 μL of 0.1% TFA in Milli-Q water three times. Elution solution (50% acetonitrile/ 0.1% TFA in Milli-Q water) was used to directly elute sample

■

RESULTS AND DISCUSSION The detailed fabrication procedure of the novel sample support is shown in the Experimental Section, as shown in Figure S1 (see Supporting Information). The outermost hydrophobic layer of the novel sample support is a fluorine-terminated SAM, and the inside one is a PMMA coating. The gap between the SAM and PMMA is bare silicon that leaves a hydrophilic area. The schematic flowchart of peptides/proteins enrichment and self-desalting process on the novel sample support is shown in 2119



Letter

Figure 1. Mass spectra of the peptides: bradykinin 1-7 fragment (M1, m/z: 756.85), angiotensin III human (M2, m/z: 931.10), and angiotensin II human (M3, m/z: 1046.18) (5 fmol μL−1 each in solution) obtained with (a) the traditional MALDI plate and (b) the novel sample support; of lysozyme (50 fmol μL−1) obtained with (c) the traditional MALDI plate and (d) the novel sample support, respectively.

proposed method is attributed to the excellent peptides/ proteins enrichment efficiency. Therefore, a series of dilution experiments ultimately were performed to demonstrate a limit of detection (LOD) for the analysis of angiotensin III human at 100 attmol μL−1 with the novel sample support, which is 2 orders of magnitude lower than that detected on the traditional MALDI plate, as shown in Figure S2 (see Supporting Information). The results clearly demonstrate a high efficiency of this novel technique in peptides/proteins enrichment. An excellent desalting approach or technique is desirable for the use of MS to obtain a satisfactory analysis.8,9,19 Since the presence of contaminants such as salts or surfactants in biological samples often result in weak or even no detectable MS signal.8,14,15 Depicted in Figure 2 are the MS spectra of 50 fmol μL−1 peptide mixture in the presence of different kinds of contaminants. Only weak signals that correspond to the three peptides can be observed by the “sweet-spot” searching on the traditional MALDI plate in the presence of 1 M NaCl, as shown in Figure 2a. Previous studies have shown that the nonvolatile salts could be disrupted with the cocrystallization of peptides and matrix, resulting in severe suppression of the MS signals.8,30,31 Nevertheless, greatly enhanced peptide signals are collected without “sweet-spot” searching on the novel sample support (Figure 2d), which indicates that the salts are effectively removed from the analyte spot and the homogeneous crystallization between the peptides and matrix is formed.27,32 The removal of salt contaminants and the homogeneous crystallization can be observed directly from the SEM images, which is proved by the EDX spectrum as shown in Figure S3 (see Supporting Information). The S/N ratios are increased from 16.43 (M1, m/z: 756.85), 65.15 (M2, m/z: 931.10), and 14.71 (M3, m/z: 1046.18) to 195.60, 108.18, and 43.75, respectively. Similarly, no peptide signals are observed in the mass spectrum due to the signal suppression effect of 100 mM NH4HCO3 on the traditional MALDI plate

Scheme 1. When the sample solution is deposited onto this support, the contact interface between the solution and the substrate is associated with wettability based on the LaplaceYoung equation,6 which results in the sample solution to be confined in the small area by the outermost hydrophobic fluorine-terminated SAM.5,9,28,29 Subsequently, in the sample drying process, the peptides/proteins are captured by the PMMA coating in the center spot through strong hydrophobic interaction between peptides/proteins and PMMA,14,15,27 while the water dissolved salt contaminations are driven into the hydrophilic silicon gap between the SAM and PMMA due to the repulsion of hydrophobic polymers.5,6,27 With the application of the matrix solutions onto the dried sample surface, the peptides/proteins and the salts are further dissolved and redistributed in hydrophobic and hydrophilic areas, respectively. Finally, the analyte−matrix cocrystallization could be formed in the center region, while the outside is filled with salts crystals. The peptides/proteins enrichment efficiency of this method is evaluated by detecting the peptide mixture including bradykinin 1-7 fragment, angiotensin II human and angiotensin III human (5 fmol μL−1 each in solution), and lysozyme protein (50 fmol μL−1) on the traditional MALDI plate and the novel sample support, respectively. As shown in Figure 1a, no assignable signal could be observed on the traditional MALDI plate for the peptides at this concentration. In contrast, the signals with excellent signal-to-noise (S/N) ratios corresponding to the three peptides in the mixture are detected using the novel sample support (Figure 1b). Similarly, the signal intensity of lysozyme (m/z: 14 400) is very weak and comparable to the noise level with a S/N ratio of 2.35 on the traditional MALDI plate (Figure 1c). Conversely, strong ion peaks of lysozyme are obtained with a great increased intensity on the novel sample support (S/N: 31.60), as shown in Figure 1d. The increase in the signal intensity as well as in the S/N ratio observed with the 2120



Letter

Figure 2. Mass spectra of the peptides: bradykinin 1-7 fragment (M1: m/z: 756.85), angiotensin III human (M2: m/z: 931.10), and angiotensin II human (M3: m/z: 1046.18) (50 fmol μL−1 each in solution) obtained in the presence of different types of salts: on the traditional MALDI plate (a) NaCl (1 M), (b) NH4HCO3 (100 mM), and (c) urea (1 M); on the novel sample support (d) NaCl (1 M), (e) NH4HCO3 (100 mM), and (f) urea (1 M).

of the novel sample support for the analysis of peptides/ proteins with MALDI MS. Furthermore, the practical application of this novel method is demonstrated by the analysis of the myoglobin digests (50 fmol μL−1). The identified peptides are listed in Table S2 (see Supporting Information). Only 7 peptide peaks are identified in the myoglobin digests with sequence coverage of 47% on the traditional MALDI plate (Figure 3a). In contrast, 14 peptide peaks are assigned to myoglobin digests with sequence coverage of 74% (Figure 3b) when the novel sample support is used. The enhanced signals could be attributed to the removal of salt contaminants and the enrichment of the peptides. On the basis of the above results, the proposed method for on-plate peptides/proteins enrichment and selfdesalting should be of great potential application in the real proteomic research.

(Figure 2b). However, after selective capture and enrichment on the novel sample support, the peaks corresponding to the three peptides become dominant in the spectrum, where the signal intensity of bradykinin 1-7 fragment (M1) is 2000 times higher than that obtained on the traditional MALDI plate (Figure 2e). In the presence of 1 M urea, no assignable peaks can be detected on the traditional MALDI plate as shown in Figure 2c, which is attributed to the formation of a thick layer of urea precipitates after solution drying that impeded seriously the UV absorption and the energy transfer from the matrix to the analytes.19 Dramatically, highly distinguishable ion peaks of the three peptides are obtained after selective enrichment and desalting on the novel sample support (Figure 2f). Overall, good spectrum-to-spectrum reproducibilities for the detection of the peptides in the mixture are obtained in the presence of different salts as shown in Table S1 (see Supporting Information) when the novel sample support is used. The relative standard deviations (RSDs) of each peptide for five time detections are less than 15%. In addition, the desalting efficiency of this novel method is further demonstrated by comparing with the commercial ZipTipC18. Figure S4 (see Supporting Information) shows that lower ion abundances of the three peptides with lower S/N ratios are obtained using the ZipTipC18 compared to the use of the novel sample support. These results demonstrate an excellent self-desalting capability

■

CONCLUSIONS In summary, a new technique has been proposed to achieve onplate simultaneous peptides/proteins enrichment and wash-free self-desalting on a novel sample support with a circle hydrophobic−hydrophilic−hydrophobic pattern. The sample solution is not only concentrated in a small region after deposition, but also the salt contaminants and the peptides/ proteins can be selectively captured in different areas in one 2121



Letter

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Z.F.Z. and Y.D.W. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (21175056) and Natural Science Foundation of Jilin Province (201015107).

■

Figure 3. Mass spectra of the myoglobin digests (50 fmol μL−1) obtained (a) on the traditional MALDI plate and (b) on the novel sample support. Peaks with ∗ are assigned to peptides from myoglobin.

step, resulting in an excellent enrichment and desalting efficiency. As a result, the detection sensitivity is improved by 2 orders of magnitude better than that from the traditional MALDI plate. At the same time, high-quality mass spectra can be obtained even with the presence of NaCl (1 M), NH4HCO3 (100 mM), or urea (1 M). The practical application of this method is further demonstrated by the successful analysis of peptides in myoglobin digests with high sequence coverage. Therefore, the novel one-step enrichment and self-desalting technique should be of great potential applications in the real proteomic research.

■

ASSOCIATED CONTENT

S Supporting Information *

Figure S-1: Schematic illustration for the preparation of the novel sample support; Figure S-2: The limit of detection of peptide (angiotensin III human, Mw = 931.10): (a) 10 fmol μL−1, with the traditional MALDI plate; (b) 100 attmol μL−1, with the novel sample support; Figure S-3: EDX/SEM images of salt-containing sample preparation on the novel sample support: (a) the salt contaminants are separated from the analytes; (b) the analyte−matrix cocrystallizations are distributed in the center polymer surface; Figure S-4: Mass spectra of the peptide mixture (50 fmol μL−1) obtained with the ZipTipC18 in the presence of: (a) NaCl (1 M), (b) NH4HCO3 (100 mM), and (c) urea (1 M); Table S-1: The spectrum-tospectrum reproducibility of peptide mixture (50 fmol μL−1) in the presence of different salts with the novel sample support. Table S-2: Peptides detected in the myoglobin digests solution using the novel sample support and the traditional MALDI plate. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

(1) Karas, M.; Bahr, U.; Giessmann, U. Mass Spectrom. Rev. 1991, 10, 335−357. (2) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269−295. (3) Seidler, J.; Zinn, N.; Boehm, M. E.; Lehmann, W. D. Proteomics 2010, 10, 634−649. (4) Peterson, D. S. Mass Spectrom. Rev. 2007, 26, 19−34. (5) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436−3442. (6) Chen, S. Y.; Li, K. I.; Yu, C. S.; Wang, J. S.; Hu, Y. C.; Lai, C. C. Anal. Chem. 2010, 82, 5951−5957. (7) Kallweit, U.; Bornsen, K. O.; Kresbach, G. M.; Widmer, H. M. Rapid Commun. Mass Spectrom. 1996, 10, 845−849. (8) Xu, Y. D.; Bruening, M. L.; Watson, J. T. Mass Spectrom. Rev. 2003, 22, 429−440. (9) Xu, Y. D.; Watson, J. T.; Bruening, M. L. Anal. Chem. 2003, 75, 185−190. (10) Chen, H. M.; Deng, C. H.; Li, Y.; Dai, Y.; Yang, P. Y.; Zhang, X. M. Adv. Mater. 2009, 21, 2200−2205. (11) Pan, C. S.; Xu, S. Y.; Zhou, H. J.; Fu, Y.; Ye, M. L.; Zou, H. F. Anal. Bioanal. Chem. 2007, 387, 193−204. (12) Qi, J. F.; Isupov, M. N.; Littlechild, J. A.; Anderson, L. E. J. Biol. Chem. 2001, 276, 35247−35252. (13) Bagshaw, R. D.; Callahan, J. W.; Mahuran, D. J. Anal. Biochem. 2000, 284, 432−435. (14) Jia, W. T.; Chen, X. H.; Lu, H. J.; Yang, P. Y. Angew. Chem., Int. Ed. 2006, 45, 3345−3349. (15) Chen, H. M.; Deng, C. H.; Zhang, X. M. Angew. Chem., Int. Ed. 2010, 122, 617−621. (16) Shen, W. W.; Xiong, H. M.; Xu, Y.; Cai, S. J.; Lu, H. J.; Yang, P. Y. Anal. Chem. 2008, 80, 6758−6763. (17) Zhang, Y. H.; Wang, X. Y.; Shan, W.; Wu, B. Y.; Fan, H. Z.; Yu, X. J.; Tang, Y.; Yang, P. Y. Angew. Chem., Int. Ed. 2005, 44, 615−617. (18) Chen, H. M.; Liu, S. S.; Li, Y.; Deng, C. H.; Zhang, X. M.; Yang, P. Y. Proteomics 2011, 11, 890−897. (19) Duan, J. C.; Wang, H.; Cheng, Q. Anal. Chem. 2010, 82, 9211− 9220. (20) Gobom, J.; Nordhoff, E.; Mirgorodskava, E.; Ekman, R.; Roepstorff, P. J. Mass Spectrom. 1999, 34, 105−116. (21) Yuan, X. L.; Desiderio, D. M. J. Mass Spectrom. 2002, 37, 512− 524. (22) Brockman, A. H.; Dodd, B. S.; Orlando, R. Anal. Chem. 1997, 69, 4716−4720. (23) Tannu, N. S.; Wu, J.; Rao, V. K.; Gadgil, H. S.; Pabst, M. J.; Gerling, I. C.; Raghow, R. Anal. Biochem. 2004, 327, 222−232. (24) Worrall, T. A.; Cotter, R. J.; Woods, A. S. Anal. Chem. 1998, 70, 750−756. (25) Zaluzec, E. J.; Gage, D. A.; Allison, J.; Watson, J. T. J. Am. Soc. Mass Spectrom. 1994, 5, 230−237. (26) Zhang, Y.; Fang, J. H.; Kuang, Y. B.; Guo, X. X.; Lu, H. J.; Yang, P. Y. Chem. Commun. 2007, 4468−4470. (27) Jia, W. T.; Wu, H. X.; Lu, H. J.; Li, N.; Zhang, Y.; Cai, R. F.; Yang, P. Y. Proteomics 2007, 7, 2497−2506. (28) Herzer, N.; Eckardt, R.; Hoeppener, S.; Schubert, U. S. Adv. Funct. Mater 2009, 19, 2777−2781. (29) Gobom, J.; Schuerenberg, M.; Mueller, M.; Theiss, D.; Lehrach, H; Nordhoff, E. Anal. Chem. 2001, 73, 434−438. (30) Xu, S. Y.; Ye, M. L.; Xu, D. K.; Li, X.; Pan, C. S.; Zou, H. F. Anal. Chem. 2006, 78, 2593−2599.

AUTHOR INFORMATION

Corresponding Author

*Address: 2699 Qianjin Street, Changchun 130012, China. Phone: 86-431-85166389. Fax: 86-431-85166389. E-mail: [email protected]. 2122



Letter

(31) Monroe, E. B.; Koszczuk, B. A.; Losh, J. L.; Jurchen, J. C.; Sweedler, J. V. Int. J. Mass Spectrom. 2007, 260, 237−242. (32) Kim, Y. S.; Hurst, G. B.; Doktycz, M. J.; Buchanan, M. V. Anal. Chem. 2001, 73, 2617−2624.

2123


Proteins

Recommend Documents