A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection

Abstract

A novel electrochemical (EC) method for fast and efficient reduction of the disulfide bonds in proteins and peptides is presented. The method does not use any chemical agents and is purely instrumental. To demonstrate the performance of the EC reactor cell online with electrospray mass spectrometry, insulin and somatostatin were used as model compounds. Efficient reduction is achieved in continuous infusion mode using an EC reactor cell with a titanium-based working electrode. Under optimized conditions, the presented method shows almost complete reduction of insulin and somatostatin. The method does not require any special sample preparation, and the EC reactor cell makes it suitable for automation. Online EC reduction followed by collision-induced dissociation fragmentation of somatostatin showed more backbone cleavages and improved sequence coverage. By adjusting the settings, the EC reaction efficiency was gradually changed from partial to full disulfide bonds reduction in α-lactalbumin, and the expected shift in charge state distribution has been demonstrated. The reduction can be controlled by adjusting the square-wave pulse, flow rate or mobile phase composition. We have shown the successful use of an EC reactor cell for fast and efficient reduction of disulfide bonds for online mass spectrometry of proteins and peptides. The possibility of online and gradual disulfide bond reduction adds a unique dimension to characterization of disulfide bonds in mid- and top-down proteomics applications.FigurePrinciple of electrochemical reduction of disulfide bonds in proteinsElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-013-7374-3) contains supplementary material, which is available to authorized users.

Comparative Effects of Ohmic, Induction Cooker, and Electric Stove Heating on Soymilk Trypsin Inhibitor Inactivation

“…The SH content after electric stove heating was slightly lower than for the induction cooker, due to the stronger thermal effect of the electric stove over the induction cooker (Table ) (Wang and Damodaran ). Several reports (Zhang and others ; Kraj and others ; Mysling and others ) have used square‐wave pulse (potentials were −1.5 V [1990 ms] and +1.0 V [1010 ms]) to efficiently reduce the SS bonds (μM/nM level) to SH. In this study, alternating current (220 V, 50 Hz), which was somewhat similar to square‐wave pulse, was used to treat 400 g of raw soymilk.…”

Characterization of cyclic peptides containing disulfide bonds

“…However, other methods have been reported that are equally applicable, including reagents such as performic acid [20], sodium sulfite [21], and tris(2-carboxyethyl)phosphine (TCEP) [16]. More recent advances include electrochemical methods [22-23]. …”

The study of electrochemistry with ambient mass spectrometry

“…In the EC-ESI-MS study, many experimental parameters that affected the disulfide-bond-reduction efficiency were identified, including square-wave potential settings, electrode material, solution composition and flow rate. One improvement for disulfidebond reduction is to use square-wave pulses rather than constant potentials [69,70]. The squarewave pulse plays an important role in avoiding electrode fouling and keeping the electrode clean for maintaining high-efficiency reduction.…”

“…The squarewave pulse plays an important role in avoiding electrode fouling and keeping the electrode clean for maintaining high-efficiency reduction. For example, using square-wave pulsing, the efficiency of disulfide reduction for insulin reduction was calculated as over 98% [69]. In addition, the most suitable electrode for disulfide reduction was found to be a Ti-based electrode.…”