Effect of Microwave Irradiation Time on Microwave-Assisted Weak Acid Protein Hydrolysis

Abstract

Horse heart myoglobin (MYG) and bovine serum albumin (BSA) were hydrolyzed by microwave-assisted weak-acid hydrolysis for 10, 20, 30, 40, 50, and 60 min using 2% formic acid (FA) at $100^{\circ}C$. Generally, the number of identified peptides increased with increasing irradiation time, indicating that the duration of microwave irradiation is linked to the efficiency of hydrolysis. For MYG, irradiation for 60 min provided the highest number of identified peptides, the greatest sequence coverage values and the highest MASCOT score values among the investigated irradiation times. Irradiation of BSA for 50 min, however, yielded a greater number of peptides than irradiation for 60 min due to the generation of miscleaved peptides after microwave irradiation for 50 min.

Introduction

Proteins can be hydrolyzed into peptides by enzyme or chemical digestion, to facilitate their characterization by mass spectrometry. Microwave irradiation is used to accelerate protein hydrolysis.¹ As an example of non-enzymatic hydrolysis, weak‑acid hydrolysis of protein (WAHP)² involves the hydrolysis of proteins in a weakly acidic solution (e.g., pH 2.0) for 2 h at 108°C.³ Microwave irradiation is reported to expedite WAHP.⁴ WAHP induces cleavage at the C-terminus, followed by the N‑terminus, of aspartic acid residues.⁵ We previously investigated the effects of temperature and acetonitrile (ACN) concentration on microwave-assisted WAHP, and found that a reduced temperature (e.g., 80 or 90°C) and 10% ACN could also be used for microwave-assisted WAHP.⁶ In this study, the effect of microwave irradiation time on the efficiency of WAHP was investigated using horse heart myoglobin (MYG) and bovine serum albumin (BSA). The proteins were subjected to microwave-assisted weak‑acid hydrolysis in 2% formic acid (FA) for 10, 20, 30, 40, 50, and 60 min at 100°C. Reducing the irradiation time would increase the throughput of WAHP processes.

 

Experimental

 

MYG, BSA, dl-dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC), and 2,5‑dihydroxybenzoic acid (DHB) were obtained from Sigma Aldrich (St. Louis, MO, USA). FA and HCl were purchased from Samchun (Gyeonggi-do, South Korea), H₃PO₄ from Oriental Chemical Industries (Seoul, South Korea), and ACN from Duksan Pharmaceutical (Gyeonggi-do, South Korea).

Stock solutions of 10 mg/mL MYG and BSA were prepared by dissolving 10 mg of MYG and BSA in 1 mL of 50 mM ABC buffer. To prepare MYG solution, 98 µL of 10-fold‑diluted MYG stock solution was mixed with 2 µL of FA. The solution was placed in a microwave oven (Rapid Enzyme Digestion System; ASTA, Gyeonggi-do, South Korea) for 10, 20, 30, 40, 50, and 60 min at a power of 100 W at 100°C.

To prepare BSA solution, 10 µL of BSA stock solution was dissolved in 90 µL of 50 mM ABC buffer and denatured by ultrasonication for 5 min, followed by sequential additions of 5 µL of 30 mg/mL DTT and 20 µL of 36 mg/mL IAA with 5 min ultrasonication after each addition. After being passed through a 30 kDa filter (Amicon Ultracentrifugation Filter 30 K; Sigma Aldrich), the BSA solution was subjected to WAH, as described above for MYG.

Matrix-associated laser desorption-ionization-time of flight mass spectrometry (MALDI‑TOF MS) analysis was performed using a Voyager DE-STR instrument (Applied Biosystems, Foster City, CA, USA), with 300 laser shots using a 337 nm nitrogen laser, 3 ns pulse length, 20 Hz pulse repetition rate, and positive ion reflectron mode.

The FindPept (https://web.expasy.org/findpept/) and PeptideMass (https://web.expasy.org/peptide_mass/) software packages were used to identify peptides with the enzyme option of microwave-assisted formic acid hydrolysis (C-term to D). Information on MYG and BSA can be found in UniProt (http://www.uniprot.org/) with the Swiss-Prot accession number/entry names P68082/MYG_HORSE and P02769/ ALBU_BOVIN, respectively.

Peptide mass fingerprinting analysis was performed using MASCOT public server (MatrixScience, MA, USA) at http://www.matrixscience.com/search_form_select.html. The m/z values of the identified monoisotopic peptide peaks for each experiment were used as data input. The search were performed against SwissProt proteome database of “Other mammalia” taxonomy, with a peptide tolerance set at 0.5 Da. The MASCOT score value was obtained from the candidate with the highest score for each analysis.

 

Results and discussion

WAH of MYG

MYG is present in the muscle tissue of vertebrates, and in almost all mammals. Its presence in the bloodstream is indicative of muscular damage, so MYG may have potential as a marker of heart attack in patients with chest pain. MYG consists of a single polypeptide chain of 153 amino acid residues and one heme group containing a shared iron atom.⁷

Figure 1 shows the mass spectra of MYG hydrolyzed in an aqueous solution of 2% FA at 100°C by microwave irradiation for 10, 20, 30, 40, 50, and 60 min. Table 1 shows the MYG peptides identified in Figure 1. Four peaks—AIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG (residues 111–154 with three miscleavages), AQGAMTKALELFRND (residues 128–142 with zero miscleavages), AQGAMTKALELFRNDIAAKYKELGFQG (residues 128–154 with one miscleavage), and IAAKYKELGFQG (residues 143–154 with zero miscleavages)—were found under all irradiation conditions. Here, the number of miscleavages was obtained by counting the number of aspartic acid (D) amino acids in the middle of the peptide sequence. Because the four peptides generated by WAH of MYG for 10 min were from the C‑terminus, this is likely the starting point of hydrolysis of the protein. Totals of 4, 6, 7, 8, 10, and 11 peptides were identified after WAH of MYG for 10, 20, 30, 40, 50, and 60 min, respectively. Both the sequence coverage values and the MASCOT scores increased with increasing irradiation time. The above observations suggest that the efficiency of MYG hydrolysis increased with increasing irradiation time up to 60 min.

 

Table 1. Peptides identified by MALDI-TOF MS in MYG hydrolyzed at 100°C by microwave irradiation in a 2% FA aqueous solution for the indicated times.

 

Figure 1. MALDI mass spectra of MYG hydrolyzed at 100 oC by microwave irradiation in a 2 % FA aqueous solution for (A) 0 min, (B) 10 min, (C) 20 min, (D) 30 min, (E) 40 min, (F) 50 min, and (G) 60 min.

 

WAH of BSA

The BSA precursor protein comprises 607 amino acids. An N-terminal 18-residue signal peptide is removed from the precursor protein upon secretion, and so the initial protein product comprises 589 amino acid residues.⁸ A further six amino acids are cleaved to yield the mature BSA protein (583 amino acids;⁹ molecular weight, ~ 66,500 Da).

 

Figure 2. MALDI mass spectra of BSA hydrolyzed at 100 oC by microwave irradiation in a 2 % FA aqueous solution for (A) 0 min, (B) 10 min, (C) 20 min, (D) 30 min, (E) 40 min, (F) 50 min, and (G) 60 min.

Figure 2 shows the mass spectra of BSA hydrolyzed in an aqueous solution of 2% FA at 100°C by microwave irradiation for 10, 20, 30, 40, 50, and 60 min. Table 1 shows the BSA peptides identified in Figure 1. Six peaks—DTHKSEIAHRFKD (residues 25–37 with one miscleavage), SPDLPKLKPD (residues 133–142 with one miscleavage), PNTLCDEFKAD (residues 143-153 with one miscleavage), RADLAKYICD (residues 280–289 with one miscleavage), KPLLEKSHCIAEVEKD (residues 304–319 with zero miscleavages), and PHACYSTVFDKLKHLVD (residues 389–405 with one miscleavage)—were found under all irradiation conditions. Unlike MYG, the initially generated BSA peptides were from random positions in the primary structure. In addition, irradiation of BSA for 50 min yielded the greatest number of BSA peptides and the highest MASCOT score. Furthermore, the highest sequence coverage was observed from the irradiation for 40 min. These observations are due to the presence of several peptides with high miscleavages such as MADCCEKQEPERNECFLSHKDDSPDLPKLKPD (residues 111-142 with four miscleavages), PHACYSTVFDKLKHLVDEPQNLIKQNCD (residues 389-405 with two miscleavages) and KCCAADDKEACFAVEGPKLVVSTQTALA (residues 580-607 with two miscleavages). These miscleaved peptides present after irradiation for 40 min or 50 min had disappeared after irradiation for 60 min. Significantly reduced background noise was observed from the MALDI mass spectra of BSA irradiated for 60 min compared to the other irradiation times, implying irradiation time of 60 min is necessary to detect BSA peptides with improved signal-to-noise ratios.

 

Table 2. Peptides identified by MALDI-TOF MS in BSA hydrolyzed at 100°C by microwave irradiation in a 2% FA aqueous solution for the indicated times.

 

Conclusion

MYG and BSA were subjected to microwave-assisted WAH for 10, 20, 30, 40, 50, and 60 min in 2% FA at 100°C. The numbers of peptides generally increased with increasing irradiation time. The highest number of peptides were identified from irradiation of MYG for 60 min; irradiation of BSA for 50 min, however, yielded a greater number of peptides than irradiation for 60 min, due to presence of several miscleaved peptides after microwave irradiation for 50 min. Conclusively, the optimum irradiation time for microwave-assisted WAH is 60 min, while a little reduced irradiation time (e.g., 40 or 50 min) could also be used as an optimum irradiation time for a certain protein such as BSA to obtain a higher sequence coverage or more peptide identification.