Can electromagnetic fields influence the structure and enzymatic digest of proteins? A critical evaluation of microwave-assisted proteomics protocols

Abstract

This study reevaluates the putative advantages of microwave-assisted tryptic digests compared to conventionally heated protocols performed at the same temperature. An initial investigation of enzyme stability in a temperature range of 37-80 °C demonstrated that trypsin activity declines sharply at temperatures above 60 °C, regardless if microwave dielectric heating or conventional heating is employed. Tryptic digests of three proteins of different size (bovine serum albumin, cytochrome c and β-casein) were thus performed at 37 °C and 50 °C using both microwave and conventional heating applying accurate internal fiber-optic probe reaction temperature measurements. The impact of the heating method on protein degradation and peptide fragment generation was analyzed by SDS-PAGE and MALDI-TOF-MS. Time-dependent tryptic digestion of the three proteins and subsequent analysis of the corresponding cleavage products by MALDI-TOF provided virtually identical results for both microwave and conventional heating. In addition, the impact of electromagnetic field strength on the tertiary structure of trypsin and BSA was evaluated by molecular mechanics calculations. These simulations revealed that the applied field in a typical laboratory microwave reactor is 3-4 orders of magnitude too low to induce conformational changes in proteins or enzymes.

Graphical abstract

Fig. 1

Identification of remaining full-length/intact BSA after a conventional (a) and microwave-assisted tryptic digest (b) performed at 50 °C for the indicated time periods by SDS-PAGE (12% gels, reducing conditions). One representative experiment (out of three) is shown.

Fig. 2

Comparison of the time-dependent BSA digestion using the PyMOL software. Tryptic peptides obtained after a 5 min heating period at 37 °C are shown in red while additional fragments generated after 16 h at 37 °C are highlighted in blue.

Fig. 3

Effect of an external electric field on the structure of BSA. RMSD refers to the measurement of the average distance between the atoms of superimposed BSA structures in the presence and in the absence of the external electric field. Small changes can be seen above 10⁷–10⁸ V/m (see Table S7 in the Supporting information for further details).

Fig. 4

Optimized structures for the BSA protein in the presence of various electric fields (blue color) in the X direction, overlapped with the structure of the native protein (red color).