Abstract
Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S–S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the “parent” disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.Graphical ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1258-z) contains supplementary material, which is available to authorized users.
Highlights
Disulfide bonds are crucial for protein structure and function [1, 2]
The characterization of protein disulfide bonds is challenging in terms of complexity and interpretation of mass spectrometry (MS) and MS/MS data
The use of a liquid chromatography (LC) separation in combination with EC-electrospray ionization (ESI)-MS is advantageous because the link between the disulfide-linked peptide and the disconnected peptides is conserved in the LC retention time dimension, facilitating the identification of the actual protein disulfide bonds
Summary
Disulfide bonds are crucial for protein structure and function [1, 2]. As such, changes in the disulfide arrangement have been associated with altered activity of proteins, such as in antibodies [3, 4] and hormones [5, 6]. The first analysis of the digests was performed with the cell switched off (CellOFF mode) to keep the disulfide bonds intact and detect the disulfide-linked peptides. Electrochemical reduction of disulfide bonds was performed using the optimized EC conditions (E1 = – 1.4 V, E2 = 0.4 V, t1 = 2000 ms, t2 = 1000 ms, and ts = 40 ms) resulting in the detection of the disconnected peptides.
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