Differential labeling of free and disulfide-bound thiol functions in proteins

Abstract

A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid chromatography/electrochemistry/mass spectrometry was used to assign the N-(2-ferroceneethyl)maleimide (FEM) labeled free cysteine functionalities in a tryptic digest mixture, whereas a precursor ion scan enables the detection of peptides with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) labeled disulfide-bound cysteine groups after reduction. Fragment spectra of the labeled peptides yield an excellent coverage of b-type and y-type ions. The ferrocene labeled cysteines were fragmented as 412 Da (FEM) and 455 Da (FMEA). These fragment masses are significantly higher than unlabeled amino acids or dipeptides and are easily detected. The position of free and disulfide-bound cysteine may therefore be assigned in an amino acid sequence. (J

Determination of glutathione and glutathione disulfide in biological samples: An in-depth review

“…Omitting the derivatization step greatly simplifies the sample preparation process and increases the sample throughput [99], but can result in excessive autoxidation and consequently erroneous results (detailed in Section 2.1). Several studies have benefited from the inclusion of derivatization [61,[100][101][102].…”

“…After its quantitative reaction and a subsequent reduction of the disulfide-bound thiols by TCEP, the newly formed thiol functionalities were reacted with the newly developed derivatizing agent ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) [101,102]. IAA was also employed for the simultaneous detection of free thiols and disulfides by means of LC-MS/MS [61].…”

Cysteine tagging for MS‐based proteomics

“…proteins with on-line electrochemical liquid chromatography (LC) MS and tryptic peptide analysis with MS/MS (Seiwert & Karst, 2007;Seiwert, Hayen, & Karst, 2008). The linkage and formation of disulfide bonds after reduction or oxidation was studied with tagging and MS to determine protein activation mechanisms (Barbirz, Jakob, & Glocker, 2000).…”

Quantification of phytochelatins and their metal(loid) complexes: critical assessment of current analytical methodology

“…Thiol 6 adds across the double bond of 20, forming the thiol conjugate 21. The iron core of the ferrocene moiety readily undergoes oxidation, from Fe 0 to Fe + , in the ionisation source of an ESI-MS [38]. Quantification of 21 is achieved by measuring the M + molecular ion, with detection limits for sulfur at the nanomolar level in the extract [38].…”

“…The iron core of the ferrocene moiety readily undergoes oxidation, from Fe 0 to Fe + , in the ionisation source of an ESI-MS [38]. Quantification of 21 is achieved by measuring the M + molecular ion, with detection limits for sulfur at the nanomolar level in the extract [38]. The adducts can easily be separated by reverse-phase chromatography and online detection of the iron by ICP-MS [39].…”