Identification of disulfide bonds in protein proteolytic degradation products using de novo-protein unique sequence tags approach

Abstract

Disulfide bonds are a form of post-translational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags), we unambiguously correlated the spectra to specific database proteins. Examination of the UStags' prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to approximately 10 kDa.

Figure 1

The flowchart of de novo-UStags approach for identification of disulfides in peptidome peptides. The dark blue segment of the blue loop-like diagram represents the UStag. Explanation of the flowchart is described in the text.

Figure 2

The example showing the de novo-UStags approach for identification of an intramolecular disulfide that links two pieces of separate sequences within the same human blood plasma antithrombin (AT) molecule. Black peaks and numbers represent the experimental measurements and red peaks and numbers represent the predictions of the assigned-peptide (same below). The m/z values labeled for y66-y62 represent those of the most abundant isotopic peaks. Explanation of the identification is described in the text.

Figure 3

The example showing the de novo-UStags approach for identification of an intermolecular disulfide that links two pieces of separate sequences originated from two human blood plasma vitronectin (VN) molecules. Explanation of the identification is described in the text.

Figure 4

The example showing the de novo-UStags approach for identification of an intramolecular disulfide within a single piece of sequence of prothrombin (FII). Explanation of the identification is described in the text.

Figure 5

Human plasma protein structures through identification of disulfide bonds in the degradation products. (A) Vitronectin dimer is formed by a Cys-Cys disulfide bond that linked the C terminal regions of the two protein molecules. (B) Antithrombin inhibitor inhibitory site degrades together with other sequence. (C) Plasminogen activation peptide degrades on the outside sequence of the disulfide bonds-linked sequence loop. (D) The protein state during degradation can be uncovered through identification of disulfide bond(s). Descriptions are detailed in the text.