Formation and reshuffling of disulfide bonds in bovine serum albumin demonstrated using tandem mass spectrometry with collision-induced and electron-transfer dissociation.

Ine Rombouts,Jan A Delcour,Marlies A Lambrecht,Peter Koehler,Katharina A Scherf,Bert Lagrain

doi:10.1038/srep12210

Abstract

Thermolysin hydrolyzates of freshly isolated, extensively stored (6 years, 6 °C, dry) and heated (60 min, 90 °C, in excess water) bovine serum albumin (BSA) samples were analyzed with liquid chromatography (LC) electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using alternating electron-transfer dissociation (ETD) and collision-induced dissociation (CID). The positions of disulfide bonds and free thiol groups in the different samples were compared to those deduced from the crystal structure of native BSA. Results revealed non-enzymatic posttranslational modifications of cysteine during isolation, extensive dry storage, and heating. Heat-induced extractability loss of BSA was linked to the impact of protein unfolding on the involvement of specific cysteine residues in intermolecular and intramolecular thiol-disulfide interchange and thiol oxidation reactions. The here developed approach holds promise for exploring disulfide bond formation and reshuffling in various proteins under conditions relevant for chemical, biochemical, pharmaceutical and food processing.

Highlights

Biological properties[4,15,16]
The approach allowed identifying twenty-one peptides with native Cys- and (Cys) or (Cys)[2] residues in serum albumin isolated from bovine blood plasma, further referred to as freshly isolated Bovine serum albumin (BSA) (Table 1)
Results demonstrated that isolation of BSA from plasma already impacts at least thirteen different Cys residues

Summary

Introduction

Biological properties[4,15,16]. Technological difficulties associated with current methodologies make that it is generally not known which Cys or cystine [(Cys)2] residues dominate protein (in)stability. The crevice in which the free SH group of native BSA is located unfolds upon heating[42,43] but the reactivity of the (Cys)[2] residues during heating remains to be investigated Against this background, the aim of this work was to monitor non-enzymatic PTMs involving Cys and (Cys)[2] during isolation, extensive dry storage and heating of isolated proteins by analysis of thermolytic digests with MS/MS with alternating ETD/CID. Locations of free SH groups and SS bonds in freshly isolated, extensively stored (6 °C, 6 years, dry) and heated (90 °C, 60 min, excess water) BSA were compared to those in native BSA as deduced from its crystal structure[44,45]. The approach can be applied to other proteins that are relevant in a chemical, biochemical, pharmaceutical or food context

Objectives

Results

Conclusion