Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is widely recognized as important in both the pathogenesis and subsequent pathophysiological sequelae of many diseases, including diabetes, cancer, and muscular dystrophy. A key target of RONS is the thiol group of protein cysteine residues. As thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins.

The most popular proteomics-based method for interrogating protein cysteine oxidation is via the alkylation of thiols using iodoacetamide chemistry, as in the OxICAT adaptation of the ICAT kit. However, this method is suboptimal for assessing the labile oxidative modification of cysteine residues as iodoacetamide chemistry has been shown to result in incomplete, non-specific, and slow alkylation of protein thiols.

Alkylation by maleimide chemistry is complete, highly selective, and rapid. It is therefore preferred to iodoacetamide. However, we find that in a standard proteomic workflow, significant hydrolysis of the maleimide adducts (succinimides) occurs with associated +16 m/z and +18 m/z peaks in the mass spectrum.

Indeed, when losses during a protein sample preparation workflow were tracked using 1D gel electrophoresis and MALDI-TOF mass spectrometry, it was found that up to 89% of the alkylated peptide can be hydrolysed during sample preparation. Gel electrophoresis alone resulted in an average 6.4% ± 3.9% (n = 3, mean ± SEM) hydrolysis; coomassie staining of gels 25% ± 6.8%, tryptic digests of gel bands 17% ± 21%; extraction of peptides from gel bands 13% ± 2%;  and biotin/streptavidin purification 30% ± 2%. The cumulative loss of signal for the peptide adduct at the expected m/z is so large that it can it to fall under the limit of detection, leading to incomplete profiling of the redoxome. Strategies to minimize succinimide hydrolysis will be presented.