Infrared-Assisted On-Plate Proteolysis for MALDI-TOF-MS Peptide

Jun 14, 2008 - In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing ...
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Anal. Chem. 2008, 80, 5640–5647

Infrared-Assisted On-Plate Proteolysis for MALDI-TOF-MS Peptide Mapping Sheng Wang, Huimin Bao, Luyan Zhang, Pengyuan Yang, and Gang Chen* School of Pharmacy, Department of Chemistry, Fudan University, Shanghai 200032, China In this report, infrared (IR)-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of matrix-assisted laser desorption/ionization (MALDI) plates under IR radiation. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c). It was demonstrated that IR radiation substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI time-of-flight mass spectrometry with sequence coverages of 55 (BSA) and 75% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. The present proteolysis strategy is simple and efficient, offering great promise for highthroughput protein identification. In the past decade, proteomics has drawn more and more research attention. One of its most important tasks is to develop efficient and rapid approaches to identifying various proteins. Protein digestion is a key procedure prior to mass spectrometry (MS) identification. It is of high importance to develop novel methods to achieve a highly efficient proteolysis for MS-based peptide mapping because the conventional in-solution digestion of proteins is time-consuming.1,2 Recently, the proteolytic enzyme, usually trypsin, was immobilized in the channels of microchips by sol-gel encapsulation,3,4 covalent linking,5 and multilayer assembly6,7 approaches to fabricate microfluidic enzymatic reactors for protein digestion. In comparison to conventional in-solution digestion, the digestion time was significantly reduced to less than 5 s because a high amount of enzyme could be immobilized in the channels. Besides microfluidic chips, trypsin was also immobilized in the inner bores * Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected]. Fax: +86-21-64187117. (1) Park, Z. Y.; Russell, D. H. Anal. Chem. 2000, 72, 2667–2670. (2) Slysz, G. W.; Lewis, D. F.; Schriemer, D. C. J. Proteome Res. 2006, 5, 1959– 1966. (3) Sakai-Kato, K.; Kato, M.; Toyooka, T. Anal. Chem. 2003, 75, 388–393. (4) Wu, H. L.; Tian, Y. P.; Liu, B. H.; Lu, H. J.; Wang, X. Y.; Zhai, J. J.; Jin, H.; Yang, P. Y.; Xu, Y. M.; Wang, H. H. J. Proteome Res. 2004, 3, 1201–1209. (5) Fan, H. Z.; Chen, G. Proteomics 2007, 7, 3445–3449. (6) Liu, Y.; Zhong, W.; Meng, S.; Kong, J. L.; Lu, H. J.; Yang, P. Y.; Girault, H. H.; Liu, B. H. Chem. Eur. J. 2006, 12, 6585–6591. (7) Wang, S.; Chen, Z.; Yang, P. Y.; Chen, G. Proteomics 2008, 8, 1785–1788.

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of fused-silica capillaries to fabricate flow-through bioreactors for proteolysis.8 In addition, a variety of trypsin-immobilized magnetic particles have been dispersed in protein solutions to carry out proteolysis with the aid of heat9 or microwaves,10,11 and the digestion time was less than 5 min. Recently, it was reported that microwaves alone could also enhance the efficiency of the conventional in-solution proteolysis significantly.12,13 The typical digestion time of microwave-assisted proteolysis was in the range of 5-20 min. More recently, ultrasonic waves were employed to reduce the digestion time of conventional in-solution proteolysis to 1 min.14 As an important form of electromagnetic wave, infrared (IR) ray has wavelengths between about 750 nm and 1 mm and has found a wide range of applications. It has been widely employed as a heat resource due to its high penetration ability. Based on its wavelength, it can be divided into near-IR (0.75-1.5 µm), middle-IR (1.5-5.6 µm), and far-IR (5.6-1000 µm) rays.15 However, we are not aware of early reports on IR-assisted proteolysis. It is of high interest to demonstrate the possibility of employing IR radiation as an energy source to enhance the efficiency of the conventional in-solution proteolysis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in protein chemistry and proteomics research for the identification of proteins. Protein samples were usually digested into peptides with proteases. Subsequently, the obtained digests were deposited on MALDI plates to perform MS measurements. To simplify the analysis process, on-plate proteolysis approaches were developed by combining digestion and spotting into one procedure.16–19 Trypsin was usually immobilized on the spots of the plate by (8) Licklider, L.; Kuhr, W. G.; Lacoy, M. P.; Keough, T.; Purdon, M. P. Anal. Chem. 1995, 67, 4170–4177. (9) Li, Y.; Xu, X. Q.; Deng, C. H.; Yang, P. Y.; Zhang, X. M. J. Proteome Res. 2007, 6, 3849–3855. (10) Lin, S.; Lin, Z. X.; Yao, G. P.; Deng, C. H.; Yang, P. Y.; Zhang, X. M. Rapid Commun. Mass Spectrom. 2007, 21, 3910–3918. (11) Chen, W. Y.; Chen, Y. C. Anal. Chem. 2007, 79, 2394–2401. (12) Lin, S. S.; Wu, C. H.; Sun, M. C.; Sun, C. M.; Ho, Y. P. J. Am. Soc. Mass Spectrom. 2005, 16, 581–588. (13) Pramanik, B. N.; Mirza, U. A.; Iing, Y. H.; Liu, Y. H.; Bartner, P. L.; Weber, P. C.; Bose, A. K. Protein Sci. 2002, 11, 2676–2687. (14) Rial-Otero, R.; Carreira, R. J.; Cordeiro, F. M.; Moro, A. J.; Santos, H. M.; Vale, G.; Moura, I.; Capelo, J. L. J. Chromatogr., A 2007, 166, 101–107. (15) Bell, C. E.;, Jr.; Taber, D. F.; Clark A. K. Organic Chemistry Laboratory with Qualitative Analysis, Standard and Microscale Experiments, 3rd ed.; Brooks/Cole-Thomson Learning: Pacific Grove, CA, 2001. (16) Harris, W. A.; Reilly, J. P. Anal. Chem. 2002, 74, 4410–4416. (17) Arnold, R. J.; Reilly, J. P. Anal. Biochem. 1999, 269, 105–112. (18) Dogruel, D.; Williams, P.; Nelson, R. W. Anal. Chem. 1995, 67, 4343– 4348. (19) Nelson, R. W.; Dogruel, D.; Krone, J. R.; Williams, P. Rapid Commun. Mass Spectrom. 1995, 9, 1380–1385. 10.1021/ac800349u CCC: $40.75  2008 American Chemical Society Published on Web 06/14/2008

adsorption16,17 or covalent linking.18,19 Protein samples were then deposited on the trypsin-immobilized spots and were allowed to digest in humidified enclosures with the aid of heat. Because a high amount of trypsin was immobilized on the plate, the typical time of the on-plate protein digestion was significantly reduced to 5-30 min. In the case of conventional in-solution tryptic digestion, the autolysis of protease would generate interfering fragments. A low weight ratio (typically 1:20-1:100) between trypsin and protein was usually employed and resulted in long digestion time (typically 12 h at 37 °C). It is a challenging task to enhance the digestion efficiency of the in-solution protein digestion that is performed directly on MALDI plates. In this work, IR radiation was employed to enhance the digestion efficiency of in-solution tryptic proteolysis on plates. Protein solutions containing trypsin were deposited on the spots of a MALDI plate that was exposed to an IR lamp to perform highefficiency proteolysis. The novel IR-assisted on-plate proteolysis approach has been coupled with MALDI-TOF-MS for the digestion and peptide mapping of bovine serum albumin (BSA) and cytochrome c (Cyt-c). The digestion time was significantly reduced to 5 min compared to 12 h for conventional in-solution digestion. The operation procedure, feasibility, and performance of IRassisted on-plate proteolysis are reported in the following sections. EXPERIMENTAL SECTION Reagent and Solutions. Acetonitrile (ACN) and ammonium bicarbonate were both purchased from Shanghai Chemical Reagent Co. (Shanghai, China). BSA, Cyt-c from horse heart, lysozyme (LYS) from chicken egg white, trypsin from bovine pancreas, trifluoroacetic acid (TFA), iodoacetamide (IAA), dithiothreitol (DTT), and R-cyano-4-hydroxycinnamic acid (CHCA) were supplied by Sigma (St. Louis, MO). Other chemicals were analytical grade. Normal human serum was kindly donated by the Clinical Laboratory of Zhongshan Hospital (Shanghai, China). Bovine skim milk (protein, g3.1% (w/w); fat, e0.3% (w/w); Bright Diary, Shanghai, China) was purchased from a local supermarket. All aqueous solutions were made up in doubly distilled water. The stock solutions (1 mg/mL) of BSA, Cyt-c, LYS, and casein (isolated from bovine milk) were prepared in doubly distilled water and were denatured in a 95 °C water bath for 15 min. IR-Assisted On-Plate Proteolysis System. The schematic diagram of the IR-assisted on-plate proteolysis system was illustrated in Supporting Information (SI), Figure 1. It consisted of an IR lamp (250 W, Shanghai Yaming Lighting Co. Ltd., Shanghai, China), a case fan, an iron case, a 192-spot MALDI stainless steel plate in a glass culture dish, and a temperature controller connected with a thermocouple. Both the IR lamp and the thermocouple were assembled in the iron case. The fan was fixed on the sidewall of the case to drive cool air inside to adjust the temperature. The case had a door and several heat elimination holes. The culture dish under the IR lamp should be as close to the sensing probe of the thermocouple as possible. The distance between the bottom surface of the IR lamp and the top surface culture dish was ∼20 cm. The temperature controller could turn on or turn off the case fan when the temperature in the case was higher or lower than 37 °C, respectively. Note that a piece of moist circular filter paper needed to be sandwiched between the plate and the bottom of the culture dish to humidify the enclosed cavity. More importantly, the water in the wet filter paper served as a

heat conductor between the plate and the glass dish so that the plate would not be overheated. Isolation of Casein from Milk. A volume of 50 mL of bovine skim milk was warmed to 40 °C in a water bath. Subsequently, 10% (v/v) acetic aqueous solution (∼2 mL) was added dropwise into the stirred milk to decrease its pH value to 4.8. The temperature was kept at ∼ 40 °C until the liquid changed from milky to almost clear. And then, the mixture solution was taken out from the water bath and was allowed to cool to room temperature (∼25 °C). The white precipitate was separated from the solution by centrifugation. After washing with ethanol, ethanol-ether (1:1, v/v), and ether successively (20 mL each), it was allowed to air-dry to obtain crude casein (∼1.7 g). IR-Assisted On-Plate Digestion of Protein. As illustrated in Figure 1, the sample solution of each protein was digested on the spot of the MALDI plate with the aid of IR radiation. Before digestion, the stock solutions of BSA, Cyt-c, and casein (isolated from bovine milk) were each diluted to 200 ng/µL with 10 mM NH4HCO3 buffer solution (pH 8.1) containing 5 ng/µL trypsin. A volume of 0.5 µL of each diluted protein solution was deposited on the spots of a MALDI plate. After the plate was enclosed in the humidified culture dish, it was exposed to IR radiation for 5 min to digest the protein samples. For comparison, BSA, Cyt-c, LYS, and casein (200 ng/µL each) in 10 mM NH4HCO3 buffer (pH 8.1) containing 5 ng/µL trypsin were also digested by using conventional in-solution proteolysis in a 37 °C water bath for 12 h. The weight ratio between trypsin and protein substrate was 1:40 for all the digestions performed in this work. Optimization of Digestion Time. LYS (200 ng/µL) in 200 µL of 10 mM NH4HCO3 buffer (pH 8.1) containing 5 ng/µL trypsin was also digested in a transparent Eppendorf tube to test the effect of digestion time on the digestion efficiency of IR-assisted proteolysis. The tube was exposed to the IR lamp in the proteolysis system (SI, Figure 1). The digestion system and conditions were the same as those for IR-assisted on-plate digestion except that the digestion was performed in an Eppendorf tube rather than on MALDI plate. During the digestion process, aliquots (10 µL) of the digestion solution were taken out from the tube at intervals of 0, 2.5, 5, 10, and 20 min. And then, each of them was immediately mixed with 5 µL of 0.5% TFA aqueous solution to stop the digestion process for subsequent MALDI-TOF-MS measurements. Measurement of the Temperature of MALDI Plate. A VC305 infrared thermometer (Shenzhen Victor Electronics, Shenzhen, China) was employed to measure the surface temperature of the MALDI plate without direct contact. Its accuracy is ±0.5 °C in the temperature range of -10 to 50 °C while the response time is less than 1 s, because shiny metal surfaces have very low emission factors and are therefore not suitable for measuring with an infrared thermometer. According to the instruction manual, the temperature of a layer of 50-µm-thick pressure-sensitive tape (2.0 cm × 2.0 cm) attached on the upper surface of a MALDI plate was assayed by using the infrared thermometer to obtain its surface temperature indirectly. The pressure-sensitive tape was made of polypropylene film with one surface coated with a layer of pressure-sensitive adhesive. It could adhere to the surface of Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Figure 1. Schematic diagram showing the process of IR-assisted on-plate proteolysis. (A) Depositing the mixture of protein and trypsin on MALDI plate; (B) enclosing the plate in a glass culture dish and exposing to IR radiation to accelerate the digestion; (C) MALDI-TOF-MS peptide mapping.

the MALDI plate closely so that thermal equilibrium could be attained quickly. Ultraviolet (UV)-Assisted On-Plate Digestion of Protein. To perform UV-assisted protein digestion, a volume of 0.5 µL of 200 ng/µL BSA or Cyt-c in 10 mM NH4HCO3 buffer solution (pH 8.1) containing 5 ng/µL trypsin was deposited on the spot of a MALDI plate. Subsequently, the plate was put in a culture dish that was covered with a layer of 10-µm-thick polyethylene film rather than a UV-opaque glass cover. A piece of moist filter paper needed to be sandwiched between the plate and the bottom of the culture dish for humidification purposes. After the dish was allowed to float on the water surface of a 37 °C water bath, it was exposed to UV radiation emitted from four TL60W/10R UV lamps (60 W, maximum emission wavelength, 365 nm; Philips) for 5 min to digest the protein samples. The distance between the lamps and the culture dish was ∼20 cm. MALDI-TOF-MS Analysis. Prior to MALDI-TOF-MS analysis, a volume of 0.5 µL of each sample solution obtained by using the in-vial digestion mentioned above was spotted on a MALDI plate. However, the present on-plate digestion obviated the spotting of the digests because digestion was directly performed on the spots of plate. After the sample solution on the plate was allowed to air-dry at room temperature, 0.5 µL of matrix solution (4 mg/mL CHCA dissolved in 50% aqueous ACN containing 0.1% TFA) was deposited on the dried sample and this was also air-dried. All MALDI-TOF-MS measurements were performed in positive ion mode using a 4700 proteomics analyzer (Applied Biosystems, Framingham, MA). The MS instrument was operated at an accelerating voltage of 20 kV. A 200-Hz pulsed Nd:YAG laser at 355 nm was used. Reflector mode was used to detect low-mass peptides while high-mass proteins were measured in linear mode. GPS Explorer software obtained from Applied Biosystems with MASCOT as a search engine and Swiss-Prot as a database was used to identify proteins based on peptide mass spectra. The missed cleavages of peptides were allowed up to 1. 5642

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RESULTS AND DISCUSSION The feasibility and performance of IR-assisted on-plate proteolysis were demonstrated by the tryptic digestion of BSA and Cyt-c in connection with MALDI-TOF-MS peptide mapping. Figure 2 shows the peptide mass fingerprints (PMFs) of the tryptic digests of 200 ng/µL BSA and 200 ng/µL Cyt-c obtained by using 5-min IR-assisted on-plate digestion. Both samples were well digested and positively identified. The identified peptide residues obtained were presented in Tables 1 and 2. It was found that 33 and 10 tryptic peptides were matched with the corresponding amino-acid sequence coverage of 55 and 75% for BSA and Cyt-c, respectively. The results indicate that 339 out of the 607 possible amino acids of BSA and 78 out of the 104 possible amino acids of Cyt-c have been identified (Table 3). In addition, the MALDI-TOF mass spectra of both digests were also measured over an extended m/z range (SI, Figure 2). No peak of their parent proteins was found, indicating the proteins in the sample solutions were digested almost completely. The insets of Figure 2 illustrate the MALDI-TOF mass spectra of the digests of BSA and Cyt-c obtained by using 5-min on-plate digestion in a dark humidified chamber at 37 °C for 5 min. In the absence of IR radiation, the protein molecules of BSA and Cyt-c were not well digested because only eight and five peptide peaks (Tables 1 and 2) were identified and the absolute peak intensities were much weaker than that exhibited in Figure 2A and B. The amino-acid sequence coverages of BSA and Cyt-c were 12 and 42%, respectively (Table 3). Moreover, the peaks of intact Cyt-c were found in the mass spectrum of the digest of Cyt-c obtained by using 5-min on-plate digestion in the dark at 37 °C when the m/z range was extended (SI, Figure 3), indicating that the protein in the sample solution was not digested completely in the absence of IR radiation. When IR radiation was employed to accelerate the on-plate digestion, the sequence coverages significantly increased from 12 to 55% and from 42 to 75% for BSA and Cyt-c, respectively (Table 3). Obviously, the efficiency of on-plate tryptic proteolysis was substantially enhanced by IR radiation.

Figure 2. MALDI-TOF mass spectra of the digests of 200 ng/µL BSA (A) and 200 ng/µL Cyt-c (B) in 10 mM NH4HCO3 buffer solution (pH 8.1) obtained by using 5-min IR-assisted on-plate digestion (trypsin/substrate ratio, 1:40; all matched peptides were marked with “*”). Also shown in the insets are the MALDI-TOF mass spectra of the digests of 200 ng/µL BSA and 200 ng/µL Cyt-c obtained by using 5-min on-plate digestion in a dark humidified chamber at 37 °C.

For comparison, the MALDI-TOF mass spectra of the digests of 200 ng/µL BSA and 200 ng/µL Cyt-c obtained by using 12-h conventional in-solution digestion were also measured (SI, Figure 4). All matched peptides were presented in Tables 1 and 2. The results indicated that 22 and 7 peptides were found to match with the amino acid sequence coverages of 37 and 75% for BSA and Cyt-c, respectively. Table 3 summarizes the MALDI-TOF-MS results of the digests obtained by using different digestion approaches. The identification results obtained by IR-assisted on-plate proteolysis were comparable to those based on conventional in-solution digestion. More importantly, the digestion time was significantly reduced from 12 h for in-solution digestion to less than 5 min for the present IR-based digestion. Because the solvent evaporation was inevitable for a long-time on-plate digestion, the effect of digestion time on the digestion efficiency of IR-assisted digestion was investigated by digesting LYS in a transparent Eppendorf tube in this work. Supporting Information, Figure 5A-E, illustrates the MALDI-TOF mass spectra of the digests of LSY obtained by digesting 200 ng/µL LYS in 10 mM NH4HCO3 buffer solution (pH 8.1, containing 5 ng/µL trypsin) with the assistance of IR radiation for 0 (A), 2.5 (B), 5 (C), 10 (D), and 20 (E) min, respectively. When the digestion time was 0 min, the protein molecules were not digested because only a few peaks appeared in the low-m/z range of the measured MALDI-TOF mass spectrum (SI, Figure 5A). Upon raising the digestion time from 2.5 to 5 min, the number of the matched peptides and the sequence coverage increased from 9

to 12 and from 47 to 61%, respectively (SI, Figure 5F, and SI, Tables 1 and 2). However, no significant increase in the sequence coverage and the number of matched peptides was observed when the digestion time was longer than 5 min although the absolute peak intensity of the peptides increased to some extent (SI, Figure 5C-E), indicating that 5 min was enough for IR-assisted tryptic digestion under the selected conditions. For comparison, LYS (200 ng/µL) was also digested by 12-h conventional in-solution digestion in an Eppendorf tube at 37 °C without the assistance of IR radiation. The obtained MALDI-TOF mass spectrum (SI, Figure 6) indicated that a total of 11 tryptic peptides were matched with the amino acid sequence coverage of 61% (SI, Tables 1 and 2). Obviously, IR radiation also significantly reduced the digestion time of the in-solution digestion from 12 h to 5 min while the identification results were identical (SI, Table 2). It is of interest to evaluate lower protein concentration to determine if the present proteolysis approach would be applicable to very small protein amounts. The mass spectra of the digests of BSA and Cyt-c (2 or 20 ng/µL each) obtained by using IRassisted on-plate digestion are shown in Figure 3 and SI, Figure 7. All matched peptides were summarized in SI, Tables 3 and 4. When the protein concentration was 20 ng/µL, the amino acid sequence coverages were determined to be 40 and 75% for BSA and Cyt-c, respectively (SI, Table 5). Upon decreasing the protein concentration to 2 ng/µL, the amino-acid sequence coverages of Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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Table 1. Identified Peptides of BSA Obtained by Using 5-min On-Plate Digestion (with and without the Assistance of IR Radiation) and 12-h Conventional In-Solution Digestion Coupled with MALDI-TOF-MSa position 25-34 29-34 66-75 76-88 89-105 123-130 131-138 161-167 161-168 168-183 169-183 198-204 221-228 223-232 229-235 242-248 264-280 286-297 298-309 310-318 310-336 347-359 360-371 361-371 361-374 402-412 413-433 421-433 437-451 438-451 469-482 469-489 499-507 508-523 524-544 529-547 549-561 569-580 581-597 588-597 a

peptide sequence

IR/on-plate

on-plate

in-solution

• •

•

a

DTHKSEIAHR SEIAHR LVNELTEFAK TCVADESHAGCEK SLHTLFGDELCKVASLR NECFLSHK DDSPDLPK YLYEIAR YLYEIARR RHPYFYAPELLYYANK HPYFYAPELLYYANK GACLLPK LRCASIQK CASIQKFGER FGERALK LSQKFPK VHKECCHGDLLECADDR YICDNQDTISSK LKECCDKPLLEK SHCIAEVEK SHCIAEVEKDAIPENLPPLTADFAEDK DAFLGSFLYEYSR RHPEYAVSVLLR HPEYAVSVLLR HPEYAVSVLLRLAK HLVDEPQNLIK QNCDQFEKLGEYGFQNALIVR LGEYGFQNALIVR KVPQVSTPTLVEVSR VPQVSTPTLVEVSR MPCTEDYLSLILNR MPCTEDYLSLILNRLCVLHEK CCTESLVNR RPCFSALTPDETYVPK AFDEKLFTFHADICTLPDTEK LFTFHADICTLPDTEKQIK QTALVELLKHKPK TVMENFVAFVDK CCAADDKEACFAVEGPK EACFAVEGPK

• • • • • •

• • • • •

• •

• • • •

• • • • • •

• •

• • • • • • • • • • • • • • • •

• •

• • • •

• •

• • • • • •

• • • • •

The matched peptides are labeled with “•”.

Table 2. Identified Peptides of Cyt-cObtained by Using 5-min On-Plate Digestion (with and without the Assistance of IR Radiation) and 12-h Conventional In-Solution Digestion Coupled with MALDI-TOF-MSa position 8-13 9-22 26-38 28-38 28-39 39-53 40-53 40-55 56-72 61-72 80-86 80-87 89-99 a

peptide sequence

IR/on-plate

KIFVQK IFVQKCAQCHTVEK HKTGPNLHGLFGR TGPNLHGLFGR TGPNLHGLFGRK KTGQAPGFTYTDANK TGQAPGFTYTDANK TGQAPGFTYTDANKNK GITWKEETLMEYLENPK EETLMEYLENPK MIFAGIK MIFAGIKK TEREDLIAYLK

•a • • •

on-plate

in-solution

• • • •

• • •

• • • • • • •

• • • •

The matched peptides are labeled with “•”.

BSA and Cyt-c were 31 and 62%, respectively. At the lower concentrations of 2 and 20 ng/µL, both proteins were positively validated. In addition, the effect of the reduction and alkylation of protein on IR-assisted on-plate digestion was also investigated. An amount 5644

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of 10 mg of DTT was dissolved in 1 mL of thermally denatured solution of BSA (1 mg/mL) that was kept in a 37 °C water bath for 1 h to reduce the disulfide bonds. Subsequently, 25 mg of IAA was added and the reaction was allowed to proceed in the dark at 37 °C for 1 h to introduce carbamidomethyl groups to the

Table 3. Summary of MALDI-TOF-MS Results of the Digests of BSA and Cyt-c Obtained by Using On-Plate Digestion (with and without the Assistance of IR Radiation) and Conventional In-Solution Digestion Coupled with MALDI-TOF-MS digestion methods IR/on-plate on-plate in-solution IR/on-plate on-plate in-solution a

protein

accession no.a

digestion time

sequence coverage, (%)

peptides matched

amino acids identified

BSA BSA BSA Cyt-c Cyt-c Cyt-c

P02769 P02769 P02769 P00004 P00004 P00004

5 min 5 min 12 h 5 min 5 min 12 h

55 12 37 75 42 75

33 8 22 10 5 7

339 78 230 78 44 78

P02769 and P00004 are the accession numbers of serum albumin precursor_bovine and cytochrome c_ horse, respectively.

Figure 3. MALDI-TOF mass spectra of the digests of 2 ng/µL BSA (A) and 2 ng/µL Cyt-c (B) in 10 mM NH4HCO3 buffer solution (pH 8.1) obtained by using 5-min IR-assisted on-plate digestion (trypsin/substrate ratio, 1:40; all matched peptides were marked with “*”).

free sulfhydryl groups. Supporting Information, Figure 8, illustrates the MALDI-TOF mass spectrum of the digest of 200 ng/ µL alkylated BSA obtained by using IR-assisted on plate digestion. All matched peptides were presented in SI, Table 6. A total of 37 peptides were matched with amino acid sequence coverage increased to 59% (SI, Table 6) compared with 55% for untreated BSA (Table 3), indicating that the reduction and alkylation steps could enhance the efficiency of the IR-assisted digestion of disulfide bond-containing proteins to some extent. The spot-to-spot reproducibility of IR-assisted on-plate digestion was examined from the simultaneous digestion of 200 ng/µL Cyt-c in 10 mM NH4HCO3 buffer solution (pH 8.1, containing 5 ng/µL trypsin) on 9 spots of the same MALDI plate. The digestion time was 5 min. The obtained 9 PMF spectra (not shown) were identical with the same sequence coverage of 75% except that the peak heights changed to some extent, indicating the satisfactory reproducibility of the present IR-assisted proteolysis approach. The effect of the irradiation time of IR ray on the surface temperature of the MALDI plate in a humidified culture dish was

illustrated in SI, Figure 9. When the irradiation times were 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min, the surface temperatures of the plate were 24.3, 28.6, 36.3, 38.8, 39.6, 40.7, 41.4, 42.5, 43.3, 44.1, and 45.1 °C, respectively. The temperature increased rapidly from 24.3 to 36.3 °C after the plate was exposed to IR radiation for 2 min. However, the temperature increases much slowly in a nearly linear manner upon increasing the irradiation time from 3 to 10 min. During the initial 5-min irradiation for IR-assisted proteolysis, the average surface temperature of the MALDI plate was 34.7 °C, which was lower than the temperature (37 °C) employed in conventional in-solution digestion. The suitability of IR-assisted on-plate proteolysis to complex proteins was demonstrated by digesting human serum and casein extracted from commercially available milk sample. Normal human serum contains 60-75 g/L proteins. The weight percentages of human serum albumin (HSA), R-1-globulin, R-2-globulin, β-globulin, and γ-globulin in the total serum protein are in the ranges of 53.3-70.5, 4.4-9.3, 6.4-10.3, 6.7-10.6, and 11-16.8%, Analytical Chemistry, Vol. 80, No. 14, July 15, 2008

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respectively.20 In this work, a sample of normal human serum was diluted in 10 mM NH4HCO3 solution (pH 8.1) containing 5 ng/µL trypsin at a ratio of 1:500 after it was denatured in a 95 °C water bath for 15 min.. Subsequently, 0.5 µL of the mixture was deposited on the spot of a MALDI plate and was allowed to digest under IR radiation for 5 min. The MALDI-TOF mass spectrum of the digest was shown in SI, Figure 10. A total of 35 peptides were found to match to HSA (SI, Table 4). Casein from bovine milk is not a single protein but consists mainly of R-S1-casein, R-S2-casein, β-casein, and κ-casein.21 The PMF of the digest of 200 ng/µL casein (isolated from bovine milk) obtained by using IR-assisted on-plate digestion was illustrated in SI, Figure 11A. A total of 39 peptides were matched (SI, Table 8). Based on their molecular weight, R-S1-casein (11 peptides), R-S2-casein (12 peptides), β-casein (10 peptides), and κ-casein (6 peptides) were positively identified by using GPS Explore Software with the integrated Mascot search-engine software. However, only nine peptide fragments were found to match when the same protein sample was digested by 5-min on-plate digestion in a dark humidified chamber at 37 °C (SI, Figure 11B). UV radiation was also employed to accelerate the on-plate tryptic digestion of BSA and Cyt-c in connection with MALDITOF-MS peptide mapping for control purposes. The PMFs of the tryptic digests of 200 ng/µL BSA and 200 ng/µL Cyt-c obtained by using UV-assisted digestion at 37 °C were illustrated in SI, Figure 12. The identified peptide residues were presented in SI, Tables 3 and 4. A total of 18 and 6 tryptic peptides from BSA and Cyt-c were found to match with the sequence coverages of 29 and 52% for BSA and Cyt-c, respectively. In comparison with the results of 5-min on-plate digestion in a dark enclosure (Table 3), the sequence coverages of BSA and Cyt-c increased from 12 to 29% and from 42 to 52%, respectively (SI, Table 5). The results indicated that UV radiation could also enhance the efficiency of on-plate tryptic proteolysis to some extent. However, the results of IRassisted on-plate digestion were much better than those of UVassisted on-plate digestion (SI, Table 5), implying that IR radiation showed higher acceleration performances toward proteolysis than higher energy photons in the UV range. Supporting Information, Figure 13, shows the MALDI-TOF mass spectrum of the digest of 200 ng/µL Cyt-c obtained by using 5-min UV-assisted on-plate digestion over an extended m/z range. Obviously, Cyt-c in the sample solution was not completely digested because the peaks of the parent protein still existed, indicating the limited efficiency of UV-assisted digestion. In addition, UV radiation was much more dangerous than IR radiation and was not easy to handle. The significantly enhanced digestion efficiency of the present IR-assisted proteolysis approach can be attributed to IR radiation. Photons in the infrared region of the electromagnetic spectrum have much less energy than photons in the visible or UV regions. They could only excite the vibrations in molecules (such as trypsin and proteins) in the modes of stretching, bending, rocking, and twisting.15 These vibrations increased the frequency of the interactions between trypsin and the protein molecules, resulting in highly efficient proteolysis. The IR-induced vibrations of proteins might also lead to more cleavage sites of proteins exposed (20) Luraschi, P.; Dea, E. D. Franzin. C. Clin. Chem. Lab. Med. 2003, 41, 782– 786. (21) Imadifon, G. I.; Farkye, N. Y.; Spanier, A. M. Crit. Rev. Food Sci. Nutr. 1997, 37, 663–689.

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to trypsin, resulting in easier cleavage of peptide chains. It might be the reason why more matched peptides were found in the peptide mass fingerprints of the digests obtained by using IRassisted digestion. CONCLUSIONS It can be concluded that IR-assisted on-plate proteolysis coupled with MALDI-TOF-MS is a promising strategy for the efficient protein digestion and peptide mapping. With the assistance of IR radiation, digestion time was substantially reduced to 5 min compared to 12 h for conventional in-solution digestion. The high sequence coverage and higher numbers of the identified peptides indicated its excellent digestion performance. The IRassisted digestion approach held great promise for rapid and highthroughput protein identification because hundreds of samples could be digested simultaneously within a short time (5 min). Another advantage of the present approach is its minimal sample consumption. It is important for the digestion of complex protein mixtures after being separated by capillary LC or capillary array LC because the volumes of eluents are usually limited. The ease, simplicity, efficiency, and low cost of the novel proteolysis approach indicate it may find further application in automated analysis of large sets of proteins. Further studies for a better understanding of IR-assisted enzymatic reactions are in progress. ACKNOWLEDGMENT This work was financially supported by NSFC (20675017 and 20405002), the 973 program of China (2007CB714500), and the 863 program of China (2007AA04Z309 and 2004AA639740). SUPPORTING INFORMATION AVAILABLE Eight tables and the following figures. SI-Figure 1. Schematic diagram of the IR-assisted on-plate proteolysis system. SI-Figure 2. MALDI-TOF mass spectra of the digests of 200 ng/µL BSA (A) and 200 ng/µL Cyt-c (B) in 10 mM NH4HCO3 solution (pH 8.1) obtained by using 5-min IR-assisted on-plate digestion over a larger m/z range (trypsin/substrate ratio, 1:40). SI-Figure 3. MALDITOF mass spectrum of the digest of 200 ng/µL Cyt-c in 10 mM NH4HCO3 solution (pH 8.1) over a larger m/z range (digestion method, on-plate digestion in a dark humidified chamber; trypsin/ substrate ratio, 1:40; digestion time, 5 min; digestion temperature, 37 °C). The peaks labeled with +1 and +2 correspond to the singly and doubly charged Cyt-c, respectively. SI-Figure 4. MALDI-TOF mass spectra of the digests of 200 ng/µL BSA (A) and 200 ng/µL Cyt-c (B) in 10 mM NH4HCO3 solution (pH 8.1) obtained by using conventional in-solution digestion (trypsin/substrate ratio, 1:40; digestion time, 12 h; digestion temperature, 37 °C; all matched peptides were marked with “*”). SI-Figure 5. MALDI-TOF mass spectra of the digests of LYS obtained by digesting 200 ng/µL LYS in 10 mM NH4HCO3 buffer solution (pH 8.1) with the assistance of IR radiation for 0 (A), 2.5 (B), 5 (C), 10 (D), and 20 (E) min (trypsin/substrate ratio, 1:40; all matched peptides were marked with “*”) and (F) effect of digestion time on the sequence coverage and the number of the identified peptides. SI-Figure 6. MALDI-TOF mass spectrum of the digest of 200 ng/µL LYS in 10 mM NH4HCO3 solution (pH 8.1) obtained by using conventional in-solution digestion (trypsin/substrate ratio, 1:40; digestion time, 12 h; digestion temperature, 37 °C; all matched peptides were marked with “*”). SI-Figure 7. MALDI-TOF mass spectra of

the digests of 20 ng/µL BSA (A) and 20 ng/µL Cyt-c (B) in 10 mM NH4HCO3 solution (pH 8.1) obtained by using 5-min IRassisted on-plate digestion (trypsin/substrate ratio, 1:40; all matched peptides were marked with “*”). SI-Figure 8. MALDITOF mass spectrum of the digest of 200 ng/µL alkylated BSA in 10 mM NH4HCO3 solution (pH 8.1) obtained by using 5-min IRassisted on-plate digestion (trypsin/substrate ratio, 1:40; all matched peptides were marked with “*”). SI-Figure 9. Effect of the irradiation time of IR radiation on the surface temperature of a stainless steel MALDI plate in a humidified culture dish. SI-Figure 10. MALDI-TOF mass spectrum of the tryptic digest of 1:500 human serum in 10 mM NH4HCO3 solution (pH 8.1, containing 5 ng/µL trypsin) obtained by using 5-min IR-assisted on-plate digestion (all matched peptides of HAS were marked with “*”). SI-Figure 11. MALDI-TOF mass spectra of the digests of 200 ng/µL casein (isolated from bovine milk) in 10 mM NH4HCO3 buffer solution (pH 8.1) obtained by using (A) 5-min IR-assisted on-plate digestion and (B) 5-min on-plate digestion in a dark

humidified chamber at 37 °C (trypsin/substrate ratio, 1:40; all matched peptides were labeled with their molecular weights and the names of their parent proteins.). SI-Figure 12. MALDI-TOF mass spectra of the digests of 200 ng/µL BSA (A) and 200 ng/µL Cyt-c (B) in 10 mM NH4HCO3 buffer solution (pH 8.1) obtained by using 5-min UV-assisted on-plate digestion (trypsin/substrate ratio, 1:40; digestion temperature, 37 °C; all matched peptides were marked with “*”). SI-Figure 13. MALDI-TOF mass spectrum of the digest of 200 ng/µL Cyt-c in 10 mM NH4HCO3 solution (pH 8.1) obtained by using 5-min UV-assisted on-plate digestion over a larger m/z range (trypsin/substrate ratio, 1:40; digestion temperature, 37 °C). The peaks labeled with +1 and +2 correspond to the singly and doubly charged Cyt-c, respectively. This materialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org. Received for review February 19, 2008. Accepted May 5, 2008. AC800349U

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