Efficient In-Gel Proteolysis Accelerated by Infrared Radiation for Protein Identification Huimin Bao,# Ting Liu,# Xian Chen, and Gang Chen* School of Pharmacy and Department of Chemistry, Fudan University, Shanghai 200032, China Received July 26, 2008
Abstract: In this report, infrared (IR) radiation was employed to enhance the efficiency of in-gel proteolysis for MS-based protein identification. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the target protein bands excised from polyacrylamide gel were cut into small pieces that were further treated in trypsin solution. Subsequently, the wet gel pieces sealed in transparent Eppendorf tubes were exposed to an IR lamp to perform IR-assisted in-gel digestion. To demonstrate the feasibility and performance of the novel digestion approach, it was employed to digest BSA and cytochrome c (Cyt-c) in polyacrylamide gels after SDSPAGE separations. The results indicated that IR radiation substantially enhanced the efficiency of in-gel proteolysis and the digestion time was significantly reduced to 5 min compared to 16 h for conventional in-gel digestion. The obtained digests were further identified by MALDI-TOF MS with improved sequence coverages. The suitability of IR-assisted in-gel proteolysis to real protein samples was demonstrated by digesting and identifying human serum albumin in gel separated from human serum by SDS-PAGE. The present proteolysis strategy is simple and efficient, offering great promise for the high-throughput protein identification in proteomics research. Keywords: Infrared • Proteolysis • Mass Spectrometry • Electrophoresis • Polyacrylamide
Introduction Proteomics has drawn more and more research attention in the past decade because it gives a much better understanding of an organism than genomics.1,2 One of its most important tasks is to develop efficient and rapid approaches to identifying a large number of proteins extracted from cells, tissue, and organisms to obtain a global perspective of the changes in protein expression.3 It is generally achieved by combining various separation techniques with mass spectrometry (MS) for the purposes of identification and quantification. In proteome research, protein mixtures are usually separated using one-dimensional or two-dimensional polyacrylamide gel electrophoresis (PAGE) into bands or spots, which are excised, digested, extracted, and finally identified by MALDI-TOF MS * To whom correspondence should be addressed.: E-mail: gangchen@ fudan.edu.cn. Fax: +86-21-64187117. # These authors contributed equally to this work. 10.1021/pr800572e CCC: $40.75
2008 American Chemical Society
Figure 1. Coomassie blue-stained polyacrylamide gels of (A) BSA, (B) Cyt-c, and (C) 1:100 diluted human serum after SDS-PAGE. Separation voltage, 120 V; separation time, ∼60 min; the amount of BSA and Cyt-c loaded on the gels, 1 µg (in 2 µL of the sample buffer for SDS-PAGE); running buffer, 25 mM Tris-192 mM glycine buffer (pH 8.3) containing 0.1%(w/v) SDS. The protein bands marked with dashed lines on the gels were excised for subsequent in-gel digestion and MS analysis.
or ESI-ion trap MS.4 However, conventional in-gel digestion is usually performed at 37 °C overnight (12-16 h) and limits the speed of large-scale protein identification.5 To date, a variety of approaches have been developed to achieve a highly efficient in-gel proteolysis. In 2003, Havlis et al. found that the reductive methylation of trypsin shifted the optimum of its catalytic activity to 50-60 °C. When the modified trypsin was used, ingel digestion can be carried out at 58 °C and the digestion time was significantly reduced from 12-16 h to 30 min without compromising the peptide yield.6 Besides higher temperature, microwaves were also employed as an efficient energy source to enhance the efficiency of conventional in-gel proteolysis and the typical digestion time was in the range of 5-10 min.4,7,8 In addition, it was demonstrated that ultrasound could accelerate in-gel proteolysis significantly. With the assistance of ultrasound, complete in-gel digestion could be attained within 1-2 min.9-11 As an important form of electromagnetic wave, infrared (IR) ray has wavelengths between 750 nm and 1 mm and has found a wide range of applications.12 In previous reports, we employed it to promote tryptic proteolysis.13 It was found that IR radiation substantially enhanced the efficiency of tryptic proteolysis and the digestion time was significantly reduced to 5 min compared to 12 h for conventional in-solution tryptic Journal of Proteome Research 2008, 7, 5339–5344 5339 Published on Web 10/23/2008
technical notes
Bao et al.
Figure 2. MALDI-TOF mass spectra of the extracted digests of 1 µg of BSA (500 ng/µL × 2 µL, A) and 1 µg Cyt-c (500 ng/µL × 2 µL, B) from gel pieces obtained by using 5-min IR-assisted in-gel digestion at 37 °C. All matched peptides were marked with “*”. Table 1. Summary of MALDI-TOF-MS Results of the Extracted Digests of 1 µg of BSA (500 ng/µL × 2 µL) and 1 µg of Cyt-c (500 ng/µL × 2 µL) Obtained by Using Infrared-Assisted and Conventional In-Gel Digestions Coupled with MALDI-TOF MS digestion methods
protein
accession no.a
digestion time
sequence coverage, (%)
peptides matched
amino acids identified
IR-assisted conventional IR-assisted conventional
BSA BSA Cyt-c Cyt-c
P02769 P02769 P00004 P00004
5 min 16 h 5 min 16 h
48 43 67 52
37 30 12 9
292 264 70 55
a
P02769 and P00004 are the accession numbers of serum albumin precursor_bovine and cytochrome c_ horse, respectively.
digestion. It is of high interest to demonstrate the possibility of employing IR radiation to enhance the efficiency of in-gel proteolysis. In this work, IR radiation was employed as an energy source to accelerate the in-gel tryptic digestion of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bands of the target proteins excised from the separation gels were cut into small pieces. After being treated with trypsin solution, the gel pieces sealed in transparent Eppendorf tubes were exposed to IR radiation to perform ingel digestion. It was found that IR radiation substantially enhanced the efficiency of in-gel proteolysis and the digestion time was significantly reduced to 5 min compared to 16 h for conventional in-solution digestion. The novel IR-assisted ingel proteolysis approach has been coupled with MALDI-TOF MS for the digestion and peptide mapping of standard proteins and human serum albumin (HSA) in gel separated from human serum by SDS-PAGE. The operation procedure, feasibility, and performance of IR-assisted in-gel proteolysis are reported in the following sections.
Experimental Section 1. Reagent and Solutions. Acetonitrile (ACN), methanol, glycine, glycerol, ammonium bicarbonate (NH4HCO3), acryla5340
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mide, N,N′-methylenebisacrylamide (bis), tris(hydroxymethyl)aminomethane (Tris), bromophenol blue, acetic acid, formic acid, and ammonium peroxodisulfate (APS) were purchased from Shanghai Chemical Reagent Company (Shanghai, China). BSA, Cyt-c from horse heart, Coomassie blue R-250, N,N,N′,N′Tetramethylethylenediamine (TEMED), sodium dodecyl sulfate (SDS), β-mercaptoethanol, trifluoroacetic acid (TFA), and R-cyano-4-hydroxycinnamic acid (CHCA) were all supplied by Sigma (St. Louis, MO). Sequencing grade TPCK-trypsin was obtained from Promega (Madison, WI). 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 (10 mg/mL) of BSA and Cyt-c were prepared in doubly distilled water. 2. Gel Electrophoresis. The instrumental setup for SDSPAGE consisted of a Mini-PROTEAN 3 electrophoresis chamber connected to an electrophoresis power supply (Bio-Rad, Hercules, CA). SDS-PAGE was carried out on self-cast polyacrylamide mini-gels (1 mm thick) using a discontinuous buffer system based on Laemmli’s method.14 The separation gel (pH 8.8) contained 12% polyacrylamide while the stacking gel (pH 6.8) contained 5% polyacrylamide. The acrylamide/bis ratio in both gels was 37.5:1. The sample buffer and the running buffer
Enhanced Proteolysis
technical notes 3. IR-Assisted In-Gel Proteolysis. The Coomassie bluestained protein bands of interest excised from the gel were cut into 1-mm3 cubes and transferred to standard Eppendorf tubes. The gel pieces of each protein band were washed with 200 µL of 50 mM NH4HCO3 containing 50% (v/v) ACN twice to remove dye, SDS, and so forth. Subsequently, 100 µL of ACN was added to dehydrate the gel pieces for 5 min. The gel pieces should shrink and look completely white. After the supernatant was discarded, the shrunk gel pieces were lyophilized for 30 min. Before digestion, 5 µL of 25 mM NH4HCO3 buffer solution (pH 8.0) containing 0.05 µg of trypsin was added to the dried gel pieces. The gel pieces were allowed to swell in the trypsin solution at 4 °C for 30 min and the extra trypsin solution was discarded with a pipet. Finally, the gel pieces were allowed to digest in the IR-assisted proteolysis system illustrated in Supporting Information (SI), Figure 1.
Figure 3. MALDI-TOF mass spectra of the extracted digests of 0.1 µg of Cyt-c (50 ng/µL × 2 µL) from gel pieces obtained by using (A) 5-min IR-assisted and (B) 16-h conventional in-gel digestion at 37 °C. All matched peptides were marked with “*”. Embedded gel images show bands of 0.1 µg of Cyt-c (in 2 µL sample buffer) excised from SDS-PAGE gels.
were 125 mM Tris-HCl buffer (pH 6.8) containing 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.025% bromophenol and 25 mM Tris-192 mM glycine buffer (pH 8.3) containing 0.1% (w/v) SDS, respectively. All solutions were freshly prepared prior to use. Before electrophoresis, the stock solution of BSA and Cyt-c was diluted to 50 or 500 ng/µL with the sample buffer. Human serum was diluted with the sample buffer at a ratio of 1:100. After these sample solutions were denatured at 95 °C for 4 min, 2 µL of each solution was loaded into the wells with a pipet using gel-loading tips. SDS-PAGE was carried out on a vertical polyacrylamide gel system at a constant voltage of 60 V until the protein bands reached the interface between stacking and separating gels. Separation was performed at a constant voltage of 120 V. The electrophoresis was stopped when the tracker dye (bromophenol) was ∼1 cm above the end of the glass plates. After being removed from the glass plates, the gel was stained for 2 h with 30% (v/v) aqueous methanol containing 0.1% (w/v) Coomassie blue R-250 and 10% (v/v) acetic acid. Proteins in the gel were fixed by acetic acid and simultaneously stained. The excess dye incorporated in the gel was removed by destaining the dyed gel with 30% (v/v) methanol containing 10% (v/v) acetic acid for 3 h. The proteins were detected as blue bands on a clear background. The images on the obtained gels were scanned by using a ScanMaker 4800 Scanner (MICROTEC, Shanghai, China). Figure 1 illustrates Coomassie blue-stained polyacrylamide gels of 1 µg of BSA, 1 µg of Cyt-c, and 2 µL of 1:100 diluted human serum after SDSPAGE separations.
The system 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 trypsin-treated gel pieces of each protein band 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. As illustrated in SI, Figure 1, the gel pieces was digested in a transparent Eppendorf tube with the aid of IR radiation. The digestion time was 5 min except when mentioned otherwise. For comparison, the gel pieces were also digested using conventional in-gel tryptic proteolysis in a 37 °C water bath for 16 h in the absence of IR radiation. Other procedures and conditions were the same as those of IR-assisted in-gel digestion. After digestion, the gel pieces of each protein band were extracted with 10 µL of 50% ACN aqueous solution containing 0.1% formic acid (w/v) with the aid of sonication for 10 min. The extracts were collected for subsequent MS analysis. 4. Optimization of Digestion Time. Four samples of polyacrylamide gel bands of Cyt-c (1 µg) after SDS-PAGE separations were digested with the assistance of IR radiation at 37 °C for 1 (A), 2.5 (B), 5 (C), and 10 (D) min, respectively. After each digestion, the obtained gel pieces were immediately extracted for subsequent MALDI-TOF-MS measurements according to the procedures mentioned above. 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 (50% (v/v) aqueous ACN containing 4 mg/mL CHCA and 0.1% (v/v) 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. Prior to MS measurement, the MS instrument was calibrated with the tryptic peptides of myoglobin in an external calibration mode. Journal of Proteome Research • Vol. 7, No. 12, 2008 5341
technical notes
Bao et al.
Figure 4. MALDI-TOF mass spectra of the extracted digests of HAS bands (Figure 1C, excised from the SDS-PAGE gels of 2 µL of 1:100 diluted human serum) obtained by using (A) 5-min IR-assisted and (B) 16-h conventional in-gel digestion at 37 °C. All matched peptides were marked with “*”.
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 in-gel proteolysis for MALDI-TOF-MS peptide mapping. Figure 2 shows peptide mass fingerprinting (PMF) spectra of the extracted digests of 1 µg of BSA (500 ng/µL × 2 µL) and 1 µg of Cyt-c (500 ng/µL × 2 µL) from gel pieces obtained by using 5-min IR-assisted in-gel digestion. Both samples were well digested and positively identified. The identified peptide residues obtained were presented in SI, Tables 1 and 2. It was found that 37 and 12 tryptic peptides were matched with the corresponding amino acid sequence coverage of 48% and 67% for BSA and Cyt-c, respectively. The results indicate that 292 out of the 607 possible amino acids of BSA and 70 out of the 104 possible amino acids of Cyt-c have been identified (Table 1). For comparison, the MALDI-TOF mass spectra of the extract digests of 1 µg of BSA (500 ng/µL × 2 µL) and 1 µg of Cyt-c (500 ng/µL × 2 µL) from gel pieces obtained by using 16-h conventional in-gel tryptic digestion in a 37 °C water bath were also measured (SI, Figure 2). All matched peptides were presented in SI, Tables 1 and 2. The results indicated that 30 and 9 peptides were found to match with the amino acid sequence coverages of 43% (identified amino acids, 264) and 52% (identified amino acids, 55) for BSA and Cyt-c, respectively. 5342
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Table 1 summarizes the MALDI-TOF-MS results of the digests obtained by using IR-assisted and conventional in-gel digestions. When IR radiation was employed to accelerate in-gel tryptic digestion, the number of the matched peptides and the sequence coverages increased from 30 to 37 and from 43% to 48% for BSA and from 9 to 12 and from 52% to 67% for Cyt-c, respectively (Table 1). The results indicate the efficiency of ingel tryptic proteolysis was substantially enhanced by IR radiation while the digestion time was significantly reduced from 16 h for conventional in-gel digestion to 5 min for the present IR-based digestion. It is of interest to evaluate lower concentration protein to determine if the present proteolysis approach would be applicable to very small protein amounts. The mass spectrum of the extracted digest of 0.1 µg of Cyt-c (50 ng/µL × 2 µL) from gel pieces obtained by using IR-assisted in-gel digestions is illustrated in Figure 3A. All matched peptides were summarized in SI, Table 3. When the concentration of Cyt-c loaded on gel was 50 ng/µL (2 µL), the amino acid sequence coverages and the number of the matched peptides were determined to be 53% (identified amino acids, 66) and 10, respectively. At the lower concentration of 50 ng/µL, Cyt-c was positively identified. However, only 7 peptides have been identified when 0.1 µg of Cyt-c (50 ng/µL × 2 µL) in gel was digested by conventional in-gel digestion with lower sequence coverage of 38% (Figure 3B and SI, Table 3). SI, Figures 3A-D illustrate the MALDI-TOF mass spectra of the extracted digests of 1 µg of Cyt-c (500 ng/µL × 2 µL) from gel pieces obtained by digesting the gel pieces with the assistance of IR radiation at 37 °C for 1 (A), 2.5 (B), 5 (C), and 10 (D) min. Upon raising the digestion time from 1 to 5 min,
technical notes
Enhanced Proteolysis the number of the matched peptides and the sequence coverage increased from 4 to 12 and from 25% to 67%, respectively. 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 (SI, Figure 3D), indicating that 5 min was enough for IR-assisted in-gel digestion under the selected conditions. Eight samples of polyacrylamide gel bands of Cyt-c (1 µg) separated by SDS-PAGE were digested by the present IRaid approach to examine its reproducibility. On the basis of the obtained 8 PMF spectra (not shown), the average sequence coverage of Cyt-c was measured to be 65% with a relative standard deviation of 4.3%, indicating the satisfactory reproducibility of IR-assisted in-gel digestion. The suitability of IR-assisted in-gel proteolysis to complex proteins was demonstrated by digesting HSA in gel separated from human serum by SDS-PAGE. Normal human serum contains 60-75 g/L of proteins.15 Figure 1C illustrates the Coomassie blue-stained polyacrylamide gel of 1:100 diluted human serum after SDS-PAGE. The darkest protein band was excised, digested, extracted, and finally identified by MALDITOF MS. The procedures and conditions could be found in the Experimental Section. The MALDI-TOF mass spectrum of the extracted digest was shown in Figure 4A. A total of 34 peptides (SI, Table 4) were found to match to HSA with sequence coverage of 48% and the darkest band in Figure 1C was identified to be Coomassie blue-stained HSA. For comparison purposes, the HAS-containing gel pieces were also digested by using conventional in-gel proteolysis in a 37 °C water bath for 16 h. Figure 4B illustrates the corresponding MALDI-TOF mass spectrum of the extract digest from the gel pieces. In the absence of IR radiation, the number of matched peptides and sequence coverage decreased to 30 and 41%, respectively, although the digestion was prolonged from 5 min to 16 h. Photons in the infrared region of electromagnetic spectrum can only excite the vibrations in molecules.12 The IR lamp used in this work could emit a continuous spectrum mainly in the wavenumber range of 600-5800 cm-1.13 To reveal the reason why IR radiation could accelerated in-gel digestion, the Fourier-transform infrared (FT-IR) spectra of protein samples were measured by using a KBr disk method on a FT-IR spectrometer (NEXUS470, NICOLET) over the wavenumber range of 4000-400 cm-1. The obtained FT-IR spectra of trypsin, 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 FTIR spectrum of BSA was illustrated in SI, Figure 4. The absorption bands near 3400, 2960, 1650, 1540, and 1395 cm-1 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 wavenumber range of the IR radiation emitted by the IR lamp. The vibrations of N-H, CdO, and C-N bonds in the in-gel proteins would lead to the vibrations of peptide bonds. These vibrations could increase the frequency of the interaction between trypsin and the peptide bonds in protein molecules, resulting in highly efficient proteolysis. More importantly, the IR-induced vibrations could also increase the energy level of cleavage sites in proteins and resulting in easier tryptic cleavage. In addition, the sequence coverage decreased upon increasing the size of protein based on the fact that the sequence
coverage of Cyt-c was much lower than that of BSA or HAS (Table 1 and SI, Table 4).
Conclusions It has been demonstrated that IR-assisted in-gel proteolysis coupled with SDS-PAGE and MALDI-TOF MS is a promising strategy for the efficient separation, digestion, and identification of proteins. The efficiency of in-gel proteolysis was significantly enhanced when IR radiation was employed as an energy source instead of a water bath. With the assistance of IR radiation, digestion time was substantially reduced to 5 min compared to 16 h for conventional in-gel tryptic digestion. The high sequence coverage and higher numbers of the identified peptides indicated its excellent digestion performance. The IR-assisted digestion approach holds great promise for rapid and high-throughput protein identification because hundreds of protein-containing gel samples can be digested under IR lamps simultaneously within a short time. 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. In addition, traditional protein gel staining and destaining methods take several hours to finish. IR radiation will be employed to speed the staining and destaining process in our future endeavors. Abbreviations: IR, infrared; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Cyt-c, cytochrome c; MALDI-TOF, 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 (20875015, 20675017 and 20405002). Supporting Information Available: Schematic diagram of the IR-assisted in-gel proteolysis system, MALDITOF mass spectra of the extracted digests of 1 µg of BSA (500 ng/µL × 2 µL) and 1 µg Cyt-c (500 ng/µL × 2 µL) from gel pieces obtained using 16-h conventional in-gel digestion at 37 °C, MALDI-TOF mass spectra of the extracted digests of 1 µg of Cyt-c (500 ng/µL × 2 µL) obtained by digesting the gel pieces with the assistance of IR radiation at 37 °C for 1, 2.5, 5, and 10 min, and FT-IR spectrum of BSA; tables of the identified peptides in the extracted digests of 1 µg of BSA (500 ng/µL × 2 µL), 1 µg of Cyt-c (500 ng/µL × 2 µL), 0.1 µg of Cyt-c (50 ng/µL × 2 µL), and HAS bands (excised from the SDS-PAGE gels of 2 µL of 1:100 diluted human serum) obtained using 5-min IR-assisted and 16-h conventional in-gel digestions coupled with MALDI-TOF MS. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) (2) (3) (4)
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PR800572E
