Weak Acid Hydrolysis of Proteins

Abstract

Weak acid hydrolysis of proteins (WAHP) is a process where a weak acid solution is used at a high temperature to hydrolyze proteins. While high-temperature solutions of strong acids result in the complete hydrolysis of proteins into individual amino acids, WAHP predominantly cleaves aspartyl residues at the C-terminus and occasionally aspartic acid residues at the N-terminus but is ineffective at room temperature. Optimal cleavage is observed after 2 h at pH 2.0 and 108 °C. Recent investigations have shown that WAHP can be expedited by exposing the digestion mixture to microwave radiation. A previous article described microwave-assisted WAHP (MAWAHP) and confirmed the identities and relative abundances of hydrolyzed peptides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in positive ion reflectron mode. The present paper describes the use of a positive ion linear mode MALDI-MS in analyses of WAH hydrolysates of myoglobin. The experimental procedure was the same as that published previously except that the mass spectra were obtained in linear mode, which is more sensitive than reflectron mode. Figure 1 shows MALDI mass spectra of myoglobin after hydrolysis in 2% formic acid. Microwave-assisted digestion (Figure 1(a)) was performed for 1 h at three different temperatures (37 °C, 50 °C, and 100 °C). Digestion was also performed in boiling water without microwave irradiation (Figure 1(b)) at incubation times of 20, 40, 60, and 120 min. Protein peaks were observed in the mass spectra of myoglobin that had been hydrolyzed by MAWAH at 37 °C and 50 °C for 1 h and in the mass spectrum of myoglobin that had been hydrolyzed in boiling water for 20 min. Supplementary Figures S1 and S2 show enlarged mass spectra from m/z 1000 to m/z 8000 with complete assignments of the MAWAH hydrosylates of myoglobin and the