In protein analysis, digestion of proteins using trypsin is an essential procedure to make small peptide fragments which are then analyzed by a mass spectrometer (MS), where overnight digestion at 37℃ is a common digestion procedure. Recently, a lot of efforts have been exerted to expedite the digestion procedure, such as using microwave,1 ultrasound,2 or pressure.3 Previously our group has investigated the temperature effect on ultrasound-assisted digestion,4 the effect of vortex-induced vibrations on the digestion efficiency,5 and pressure-assisted digestion using a syringe.6 In a continuation of our study on protein digestion efficiency, the current study investigates the effectiveness of microwave-assisted protein digestion. Recently, microwave-assisted protein digestion7-9 is getting popular since microwave irradiation can significantly decrease digestion time, making the entire sample preparation fast and simple. Here we are investigating the effectiveness of microwave-assisted protein digestion in different well positions of a 96well microplate. A microwave oven was utilized to perform enzymatic digestion of a protein in 10 different well positions of a 96-well microplate. The digestion efficiencies of the samples in different well positions were compared by utilizing a matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) MS.
Trifluoroacetic acid (TFA, ≥ 98%), formic acid, α-Cyano4-hydroxy-cinnamic acid, dithiothreitol (DTT, ≥ 99%), iodoacetamide (IAA, 99%), bovine serum albumin (BSA, ≥ 96%), and acetonitrile (ACN) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sequence grade trypsin was obtained from Promega (Cat. No. v5113; Madison, WI, USA).
Protein stock solutions were prepared by dissolving 10 mg BSA in 1.0 mL of 50 mM ammonium bicarbonate buffer solution. The denaturation of BSA was performed according to established procedures.6 Briefly, 10 mg BSA was dissolved in 1 mL of 6 M guanidine·HCl in 50 mM ammonium bicarbonate buffer (pH 8.3). Then, 10 mM DTT was added to the BSA solution. After reduction for 30 min at 56℃, 40mM IAA was added to the solution for alkylation in the dark for 30 min at room temperature, and subsequently the solution was desalted on a commercial YM-10 desalting spin column. The volume of the regained solution was adjusted to 1.0 mL using a micropipette. Then, 10 µL of the BSA solutions (10 mg/mL) was mixed with 4.0 µL of 0.5 µg/µL trypsin and 86 µL of 50 mM ammonium bicarbonate buffer (1:50 trypsin/protein ratio) in a 96-well plate. The final concentration of BSA sample solution was approximately 15 pmol/µL.
To study the reproducibility of microwave-assisted digestion under different positions, tryptic digestion of BSA was performed in 10 random well positions (A1, A12, B9, C3, D6, E2, E10, G7, H1, and H12) of a 96-well microplate for 10 min at 37℃ with 400 W using a Microwave Digestion System (HST Rapid Enzyme Digestion System) from Hudson Surface Technology (Fort Lee, NJ, USA) as instructed in the user’s manual. Figure 1 shows the 96-well microplate with sample locations used for this study. After the digestion, 1 µL formic acid was added to each well to stop the reaction. For comparison, tryptic digestion of BSA was also performed using conventional overnight digestion at 37℃.
Figure 1.Sample locations in a 96-well microplate used for the current microwave-assisted digestion.
To prepare sample spots for MALDI-TOF MS analysis, 2 µL of the sample solution was loaded onto a MALDI plate and dried. Then, 2 µL of a CHCA matrix solution consisting of 10 mg CHCA dissolved in 1 mL of water/ACN (50:50, v/v) containing 0.1% TFA was loaded onto each sample layer, and the digested protein was confirmed by MALDI-TOF MS. The mass spectrometer (Ultraflextreme time-of-flight mass spectrometer made by Bruker Daltonics, Germany) equipped with a 355-nm Nd:YaG laser was used to obtain mass spectra. Total shots were accumulated and mass spectra were obtained from m/z 400-5000 in a positive ion reflectron mode.
The identification of digested peptides was done automated database searching of peak list files using the MASCOT search program (http://www.matrixscience.com). Database searches were restricted to the protein sequence of the corresponding protein downloaded from the SwissProt database. The following search parameters were selected for all database searching: enzyme, trypsin; missed cleavages, 5; peptide tolerance, ± 0.5 Da; mass values, MH+; fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M). The search results, including unique peptide sequences, sequence coverage, calculated molecular mass of the peptide, were extracted.
Results and Discussion
Digestion efficiency of microwave-assisted digestion in various locations
In the current study, we are investigating the effectiveness of microwave-assisted protein digestion in different well positions of a 96-well microplate. Figure 1 shows the 10 random well positions where microwave-assisted digestion was performed (A1, A12, B9, C3, D6, E2, E10, G7, H1, and H12) on a 96-well microplate. Figure 2 displays the mass spectra of microwave-assisted digestion of BSA from the 10 well positions along with the mass spectrum of conventional overnight digestion, where similar patterns were observed for the former 10 mass spectra. In order to obtain detailed information, the peptide peaks were selected and searched using the MASCOT search program to identify the peaks. Supplementary table S1 exhibits the comprehensive information of the identified peptides from microwave-assisted digestion and conventional overnight digestion. Table 1 shows the summary of the comparison of digestion efficiency between microwave-assisted digestion at different well-positions and conventional overnight digestion.
As shown in Table 1, the sequence coverages of BSA from microwave-assisted digestion obtained from most well positions of the 96-well microplate were observed higher than that from conventional overnight digestion where the average sequence coverages from microwave-assisted digestions was 71.7%, while the sequence coverage from overnight digestion was 63%. The ratios of missed cleavages from the samples of the 10 well positions of the 96-well microplate (~53.3%) were all higher than the ratio from overnight digestion (42.1%). The increased sequence coverages from microwave-assisted digestion compared to the overnight digestion are believed to be due to the increased miscleavages. Miscleaved peptides contain additional basic amino acids, which assists the ionization of the peptides.10, 11
Comparison of miscleavage ratio between different digestion methods
The average miscleavage ratio of the current ultrasound-assisted digestions was 53.3%, while the miscleavage ratio of conventional overnight digestion was 42.1%. The miscleavage ratios from so-called assisted digestions were always observed to be higher than those from conventional overnight digestion, such as pressure-assisted digestion using a syringe for 30 min at 37℃ and 6 atm (43.2% vs. 24.2%),6 vortex-assisted digestion for 1hr at 37℃ with 2,500 rpm (53.3% vs. 28.6%),5 and ultrasound-assisted digestion at 55℃ for 5 min (62.5% vs. 28.6%)4 for the analysis of BSA using MALDI-TOF MS. All of the above mentioned assist-digestion methods definitely expedite digestion process. However, the reduced digestion time is not long enough to reach a digestion status obtained from overnight digestion. Since the existence of miscleaved peptides generally provides more information of a protein by increasing the sequence coverage, the incomplete digestion may be more informative than complete digestion. The point is then to generate reproducible miscleaved peptides. The current analysis shows that the microwave-assisted digestion can provide reproducible digestion results. Reproducible digestion results can be useful for the quantitative analysis of proteins. The small variation of the sequence coverage or the number of miscleaved peptides will be further reduced if some sort of shaking device is used during the microwave-assisted digestion process.
Figure 2.MALDI-TOF mass spectra of tryptically digested peptides of BSA produced by microwave-assisted digestion in 10 different well positions of a 96-well plate (a-j) and by conventional overnight digestion (k). Around 30 pmol was used for each mass spectrum. The exact sample locations are shown in Figure 1.
Table 1.a)The ratio was calculated using the number of miscleaved peptides divided by the number of identified peptides.
Microwave-assisted digestion for 10 min at 37℃ with 400 W was performed for BSA in 10 different positions of a 96-well plate, where similar increased sequence coverage (~70%) was observed for the 10 samples compared to conventional overnight digestion whose sequence coverage was 63%. In terms of miscleavage, higher miscleavage ratios were observed from microwave-assisted digestion than conventional overnight digestion. The reproducible results from the 10 microwave-assisted digestions from different locations demonstrate that microwave can be successfully utilized for the quantitative analysis of protein samples.
Additional supporting information may be found in the online version of this article.