Efficient Chymotryptic Proteolysis Enhanced by Infrared Radiation for Peptide Mapping Sheng Wang,# Ting Liu,# Luyan Zhang, and Gang Chen* School of Pharmacy and Department of Chemistry, Fudan University, Shanghai 200032, China Received June 28, 2008
Abstract: Infrared (IR) radiation was employed to enhance the efficiency of chymotryptic proteolysis for peptide mapping in this work. Protein solutions containing chymotrypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 °C. BSA and cytochrome c (Cyt-c) were digested by IR-assisted chymotryptic proteolysis to demonstrate the feasibility and performance of the novel digestion approach and the digestion time was significantly reduced to 5 min. The obtained digests were further identified by MALDI-TOF MS with the sequence coverages that were comparable to those obtained by using conventional in-solution digestion. The suitability of IR-assisted chymotryptic proteolysis to complex proteins was demonstrated by digesting human serum. The present proteolysis strategy is simple and efficient, offering great promise for highthroughput protein identification. Keywords: Infrared • Proteolysis • Mass Spectrometry • Protein • Chymotrypsin
Introduction Proteomics is the large-scale study of proteins, particularly their structures and functions. As one of the fastest developing areas in biological research, it has drawn more and more research attention.1,2 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 conventional insolution digestion is time-consuming.3,4 A variety of approaches have been developed to enhance the efficiency of proteolysis. Trypsin has been immobilized in the channels of microchips by sol-gel encapsulation,5,6 covalent linking7 and multilayer assembly8,9 approaches to fabricate microfluidic enzymatic reactors for protein digestion. Because a high amount of enzyme could be immobilized in the channels, the digestion time was significantly reduced to less than 5 s compared to 12 h for conventional in-solution digestion. Besides microfluidic chips, trypsin and chymotrypsin have also been immobilized in the inner bores of fused silica
capillaries to fabricate flow-through bioreactors for proteolysis.10-12 In addition, a variety of trypsin-immobilized magnetic particles have been dispersed in protein solutions to carry out proteolysis with the aid of heat13 or microwaves14,15 and the digestion time was less than 5 min. Conventional in-solution digestion has been commonly used in proteolysis. However, the autolysis of protease would generate interfering fragments. A low weight ratio (typically 1:20 to 1:100) between protease and protein was usually employed and resulted in long digestion time (typically 12 h at 37 °C).4 It is a challenging task to enhance the digestion efficiency of conventional in-solution protein digestion. Recently, microwaves were employed to enhance the efficiency of conventional insolution proteolysis significantly.16,17 The typical digestion time of microwave-assisted proteolysis was in the range of 5-20 min. Moreover, it was reported that ultrasonic waves could also accelerate the conventional in-solution digestion of proteins and the digestion time was reduced to 1 min.18 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.19 In a previous report, we have employed IR radiation as an energy source to promote tryptic proteolysis for the first time and the digestion could complete within 5-10 min.20 R-Chymotrypsin is another commonly used protease that selectively catalyzes the hydrolysis of peptide bonds on the C-terminal side of tyrosine, phenylalanine, tryptophan, and leucine. The typical time of in-solution chymotryptic proteolysis is in the range of 12-24 h.21,22 It is of high interest to demonstrate the possibility of employing IR radiation as an energy source to enhance the efficiency of in-solution chymotryptic proteolysis. In this work, IR radiation was employed to enhance the digestion efficiency of in-solution chymotryptic proteolysis. Protein solutions containing chymotrypsin in sealed transparent Eppendorf tubes were allowed to be exposed to IR radiation to perform high efficient proteolysis. The novel IR-assisted chymotryptic proteolysis approach has been coupled with MALDI-TOF MS for the digestion and peptide mapping of 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 IR-assisted chymotryptic proteolysis are reported in the following sections.
Experimental Section * To whom correspondence should be addressed. E-mail: gangchen@ fudan.edu.cn. Fax: +86-21-64187117. # These author contributed equally to this work. 10.1021/pr800476s CCC: $40.75
2008 American Chemical Society
1. Reagent and Solutions. Acetonitrile (ACN) and ammonium bicarbonate (NH4HCO3) were both purchased from Journal of Proteome Research 2008, 7, 5049–5054 5049 Published on Web 10/18/2008
technical notes
Figure 1. Schematic diagram of the IR-assisted proteolysis system.
Shanghai Chemical Reagent Company (Shanghai, China). BSA, Cyt-c from horse heart, R-chymotrypsin from bovine pancreas, trifluoroacetic acid (TFA), and R-cyano-4-hydroxycinnamic acid (CHCA) were all 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). All aqueous solutions were made up in doubly distilled water. The stock solutions (1 mg/mL) of BSA and Cyt-c were prepared in doubly distilled water and were denatured in a 95 °C water bath for 15 min. 2. IR-Assisted Proteolysis System. The schematic diagram of the IR-assisted proteolysis system was illustrated in Figure 1. It consists of an IR lamp (250 W, Shanghai Yaming Lighting Co. Ltd., Shanghai, China), a case fan, a temperature controller connected with a thermocouple, and an iron case. Both the IR lamp and the thermocouple were assembled in the iron case. The case fan was fixed on the sidewall of the case to drive cool air inside to adjust the temperature. The iron case has a door and several heat elimination holes. The transparent Eppendorf tubes containing protein samples 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 Eppendorf tubes was approximately 20 cm. The temperature controller could turn on or turn off the case fan when the temperate in the case was higher or lower than 37 °C, respectively. Note that the tubes should be sealed with caps during the IR-assisted digestion. 3. IR-Assisted Proteolysis. As illustrated in Figure 1, the sample solution of each protein was digested in a transparent Eppendorf tube with the aid of IR radiation. Before digestion, the stock solutions of BSA and Cyt-c were each diluted to 20 or 200 ng/µL with 10 mM NH4HCO3 buffer solution (pH 8.1) containing 0.5 or 5 ng/µL chymotrypsin, respectively. The digestion time was 5 min except mentioned otherwise. For comparison, BSA and Cyt-c (200 ng/µL each) in 10 mM NH4HCO3 buffer (pH 8.0) were also digested by using conventional in-solution chymotryptic proteolysis in a 37 °C water bath for 12 h. The weight ratio between chymotrypsin and protein substrate was 1:40. 4. Optimization of Digestion Time. Cyt-c (200 ng/µL) in 200 µL of 10 mM NH4HCO3 buffer (pH 8.0) containing 5 ng/µL chymotrypsin was also digested in a transparent Eppendorf tube to test the effect of digestion time on the digestion efficiency of IR-assisted proteolysis. During the digestion process, aliquots (10 µL) of the digestion solution were taken out from the tube at intervals of 1, 2.5, 5, 10, and 20 min. And 5050
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Wang et al. 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. 5. MALDI-TOF-MS Analysis. Prior to MALDI-TOF-MS analysis, a volume of 0.5 µL of each sample solution was spotted on a MALDI 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. Reflectron 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 search was done based on the monoisotopic MH+ mass values of peptides. The peptide mass tolerance was set to (100 ppm and the missed cleavages of peptides were allowed up to 1.
Results and Discussion In this work, IR radiation was employed to enhance the efficiency of chymotryptic proteolysis for MALDI-TOF-MS peptide mapping. Panels A and C of Figure 2 show the peptide mass fingerprints (PMFs) of the chymotryptic digests of 200 ng/µL BSA (0.5 µL, 1.52 pmol) and 200 ng/µL Cyt-c (0.5 µL, 8.07 pmol) obtained using 5-min IR-assisted 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 29 and 13 chymotryptic peptides were matched with the corresponding amino-acid sequence coverage of 41% and 75% for BSA and Cyt-c, respectively. The results indicate that 254 out of the 607 possible amino acids of BSA and 79 out of the 104 possible amino acids of Cyt-c have been identified (Table 3). Supporting Information (SI), Figure 1 illustrates the MALDITOF mass spectra of the digests of 200 ng/µL BSA (0.5 µL, 1.52 pmol) and 200 ng/µL Cyt-c (0.5 µL, 8.07 pmol) obtained using 5-min in-solution 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 11 and 7 peptide peaks (Tables 1 and 2) were identified and the absolute peak intensities were weaker than that exhibited in Figure 2A,C. The amino acid sequence coverages of BSA and Cyt-c were 11% and 56%, respectively (Table 3). When the digestion was accelerated by IR radiation, the sequence coverages significantly increased from 11% to 42% and from 56% to 75% for BSA and Cyt-c, respectively (Table 3). The results indicated the efficiency of in-solution chymotryptic proteolysis was substantially enhanced by IR radiation. For comparison, the MALDI-TOF-MS mass spectra of the digests of 200 ng/µL BSA (0.5 µL, 1.52 pmol) and 200 ng/µL Cyt-c (0.5 µL, 8.07 pmol) obtained using 12-h conventional insolution chymotryptic digestion were also measured (Supporting Information, Figure 2). All matched peptides were presented in Tables 1 and 2. The results indicated that 24 and 11 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
Efficient Chymotryptic Proteolysis
technical notes
Figure 2. MALDI-TOF mass spectra of the chymotryptic digests of 200 ng/µL BSA (A, 0.5 µL), 20 ng/µL BSA (B, 0.5 µL, 0.152 pmol), 200 ng/µL Cyt-c (C, 0.5 µL, 8.07 pmol), and 20 ng/µL Cyt-c (D, 0.5 µL, 0.807 pmol) in 10 mM NH4HCO3 buffer solution (pH 8.0) obtained using 5-min IR-assisted digestion (chymotrypsin/substrate ratio, 1:40; digestion temperature, 37 °C; all matched peptides were marked with “*”).
results obtained by IR-assisted 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. 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 20 ng/µL BSA (0.5 µL, 0.152 pmol) and 20 ng/µL Cyt-c (0.5 µL, 0.807 pmol) obtained using IR-assisted chymotryptic digestion are shown in Figure 2B,D. All matched peptides were summarized in Supporting Information, Tables 1 and 2. When the protein concentration was 20 ng/µL, the amino acid sequence coverages were determined to be 38% (identified amino acids, 234) and 73% (identified amino acids, 76) for BSA and Cyt-c, respectively. At the lower concentration of 20 ng/ µL, both proteins were positively identified. Figure 3A,E illustrates the MALDI-TOF mass spectra of the digests of Cyt-c (0.5 µL, 8.07 pmol) obtained by digesting 200 ng/µL Cyt-c in 10 mM NH4HCO3 buffer solution (pH 8.0, containing 5 ng/µL chymotrypsin) with the assistance of IR radiation for 1 (A), 2.5 (B), 5 (C), 10 (D), and 20 (E) min. Upon raising the digestion time from 1 to 5 min, the number of the matched peptides and the sequence coverage increased from 7 to 13 and from 59% to 75%, respectively (Figure 3F and Supporting Information, Table 3). 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 (Figures 3C,E), indicating that 5 min was enough for IRassisted chymotryptic digestion under the selected conditions. The reproducibility of the present method was examined from a series of 7 repetitive digestions of a 200 ng/µL Cyt-c.
The obtained 7 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 IR-assisted chymotryptic proteolysis. The suitability of IR-assisted chymotryptic proteolysis to complex proteins was demonstrated by digesting human serum. Normal human serum contains 60-75 g/L of proteins. The weight percentages of human serum albumin (HSA), R-1globulin, 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%, respectively.23 In this work, a sample of normal human serum was diluted in 10 mM NH4HCO3 solution (pH 8.0) containing 5 ng/µL chymotrypsin at a ratio of 1:500 after it was denatured in a 95 °C water bath for 15 min. Subsequently, the mixture solution was allowed to digest under IR radiation for 5 min. The MALDI-TOF mass spectrum of the digest was shown in Figure 4. A total of 26 peptides (Supporting Information, Table 4) were found to match to HSA by using GPS Explore Software with the integrated Mascot search-engine software. The significantly enhanced digestion efficiency of the present proteolysis approach can be attributed to IR radiation. Photons in the infrared region of electromagnetic spectrum can only excite the vibrations in molecules (such as chymotrypsin and proteins) in the modes of stretching, bending, rocking, and twisting.19 As shown in Supporting Information, Figure 3, the IR lamp used in this work could emit a continuous spectrum mainly in the wavenumber range of 600-5800 cm-1 (corresponding wavelength range, 16.66-1.72 µm).24 The Fouriertransform infrared (FT-IR) spectra of protein samples were measured using a KBr disk method on a FT-IR spectrometer (NEXUS470, NICOLET) over the wavenumber range of 4000-400 Journal of Proteome Research • Vol. 7, No. 11, 2008 5051
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Wang et al.
Table 1. Identified Peptides in the Chymotryptic Digests of 200 ng/µL BSA (0.5 µL, 1.52 pmol) Obtained Using 5-min Infrared-Assisted Digestion and Conventional In-Solution Digestion (5 min and 12 h) Coupled with MALDI-TOF MS
a
position
peptide sequence
IR-assisted
36-43 49-54 55-60 56-66 61-70 127-139 140-150 163-171 164-171 181-188 189-201 214-229 222-229 235-242 243-251 243-257 262-273 333-342 358-364 358-369 371-376 377-393 381-393 411-418 427-434 435-453 505-511 505-514 533-555 557-567 568-574 592-606 599-606
KDLGEEQF IAFSQY LQQCPF QQCPFDEHVKL DEHVKLVNEL LSHKDDSPDLPKL KPDPNTLCDEF YEIARRHPY EIARRHPY ANKYNGVF QDCCQAEDKGACL ASSARQRLRCASIQKF RCASIQKF KAWSVARL SQKFPKAEF SQKFPKAEFVEVTKL TKVHKECCHGDL AEDKDVCKNY SRRHPEY SRRHPEYAVSVL RLAKEY EATLEECCAKDDPHACY EECCAKDDPHACY IKQNCDQF QNALIVRY TRKVPQVSTPTLVEVSRSL VNRRPCF VNRRPCFSAL HADICTLPDTEKQIKKQTALVEL KHKPKATEEQL KTVMENF AVEGPKLVVSTQTAL VVSTQTAL
•a • • • •
in-solution/5 min
in-solution/12 h
• • •
• • • •
• • •
• • • • • • • • • • • • • • • • • • •
•
• • • • • • •
• •
• • •
• • • • • • •
• • • •
• • • • •
•
The matched peptides are labeled with “•”.
Table 2. Identified Peptides in the Chymotryptic Digests of 200 ng/µL Cyt-c (0.5 µL, 8.07 pmol) Obtained Using 5-min Infrared-Assisted Digestion and Conventional In-Solution Digestion (5 min and 12 h) Coupled with MALDI-TOF MS
a
position
peptide sequence
IR-assisted
1-10 36-46 37-46 37-48 47-59 49-59 49-64 60-67 68-74 69-82 75-82 75-94 83-97 98-104
GDVEKGKKIF FGRKTGQAPGF GRKTGQAPGF GRKTGQAPGFTY TYTDANKNKGITW TDANKNKGITW TDANKNKGITWKEETL KEETLMEY LENPKKY ENPKKYIPGTKMIF IPGTKMIF IPGTKMIFAGIKKKTEREDL AGIKKKTEREDLIAY LKKATNE
•a • • • • • • • •
in-solution/12 h
• • •
• • • •
•
• • •
•
• •
• •
• •
The matched peptides are labeled with “•”.
cm-1. The obtained FT-IR spectra of chymotrypsin, BSA, and Cyt-c were identical because these proteins consisted of the “residues” of 20 proteinogenic amino acids linked by peptide bonds (-CO-NH-). The FT-IR spectrum of BSA was illustrated in Supporting Information, Figure 4. The absorption bands near 3400, 2960, 1650, 1540, and 1395 cm-l were assigned to the vibrations of N-H (stretching), C-H (stretching), CdO (stretching), N-H (bending), and C-N (stretching), respectively. Obviously, the wavenumbers of these peaks fell into the 5052
• • • •
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wavenumber range of the IR radiation emitted by the IR lamp. The vibrations of N-H, CdO, and C-N bonds in both proteins would lead to the vibrations of peptide bonds. These vibrations could increase the frequency of the interaction between chymotrypsin and the peptide bonds in protein molecules, resulting in highly efficient proteolysis. In addition, the IRinduced vibrations of the chains in proteins might also lead to more cleavage sites exposed to chymotrypsin, resulting in easier cleavage of peptide bonds. It might be the reason why there
technical notes
Efficient Chymotryptic Proteolysis
Table 3. Summary of MALDI-TOF-MS Results of the Chymotryptic Digests of 200 ng/µL BSA (0.5 µL, 1.52 pmol) and Cyt-c (0.5 µL, 8.07 pmol) Obtained Using Infrared-Assisted Digestion and Conventional In-Solution Digestion Coupled with MALDI-TOF MS digestion methods
protein
accession no.a
digestion time
IR-assisted in-solutionn in-solution IR-assisted in-solution in-solution
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
a
sequence coverage, (%)
peptides matched
amino acids identified
41 11 37 75 56 75
29 11 24 13 7 11
254 72 228 79 59 78
P02769 and P00004 are the accession numbers of serum albumin precursor_bovine and horse cytochrome c respectively.
Figure 3. MALDI-TOF mass spectra of the chymotryptic digests of 200 ng/µL Cyt-c (0.5 µL, 8.07 pmol) in 10 mM NH4HCO3 solution (pH 8.0) obtained using IR-assisted digestion for (A) 1, (B) 2.5, (C) 5, (D) 10, and (E) 20 min. Other conditions, as in Figure 2.
were more matched peptides in the PMF spectra of the digests obtained by using IR-assisted digestion.
Conclusions It has been demonstrated that IR-assisted chymotryptic 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 chymotryptic digestion. The high sequence coverage and higher numbers of the identified peptides indicated its excellent digestion performance. The IR-assisted digestion approach held great promise for rapid and high-throughput protein identification because hundreds of samples could be digested under IR lamps simultaneously within a short time (5 min). The ease, simplicity, efficiency, and low cost of the novel proteolysis approach indicate great promise for the highthroughput protein identification. In addition, IR-assisted Journal of Proteome Research • Vol. 7, No. 11, 2008 5053
technical notes
Wang et al. serum obtained using 5-min IR-assisted chymotryptic digestion coupled with MALDI-TOF MS. This material is available free of charge via the Internet at http://pubs.acs.org.
References
Figure 4. MALDI-TOF mass spectrum of the chymotryptic digest of 1:500 human serum in 10 mM NH4HCO3 solution (pH 8.0, containing 5 ng/µL chymotrypsin) obtained using 5-min IRassisted digestion (all matched peptides of HAS were marked with “*”).
chymotryptic proteolysis will find more applications in proteomics if it is directly performed on the spots of MALDI plate to digest many protein samples simultaneously. Abbreviations: IR, infrared; Cyt-c, cytochrome c; MALDITOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; PMF, peptide mass fingerprinting.
Acknowledgment. This work was financially supported by the 863 Program of China (2007AA04Z309 and 2004AA639740), National Key Technology R&D Program (2006BAI19B02), and NSFC (20675017 and 20405002). Supporting Information Available: MALDI-TOF mass spectra of the chymotryptic digests of 200 ng/µL BSA and 200 ng/µL Cyt-c in 10 mM NH4HCO3 solution (pH 8.0) obtained using 5 min and conventional in-solution digestion, figure of spectral power distribution of the IR lamp used in this work, and FT-IR spectra of BSA; tables of identified peptides in the digest of 20 ng/µL BSA and 20 ng/µL Cyt-c obtained using 5-min IR-assisted chymotryptic digestion coupled with MALDITOF MS, identified peptides in the digests of 200 ng/µL Cyt-c obtained using infrared-assisted chymotryptic digestion coupled with MALDI-TOF MS, and identified peptides of HAS in human
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PR800476S
