Download Modifications of redox-active cysteines occurring during sample

Modifications of redox-active cysteines occurring during sample
preparation
Malte Bayer* and Simone König
Core Unit Proteomics, Interdisciplinary Center for Clinical Research,
University of Münster, Germany
*Corresponding author: [email protected]
Phosphorylation is an important posttranslational protein modification. As the mass
difference to sulfonation is only 9.5 mDa, it may be difficult to distinguish these two
protein modifications with mass spectrometers of lower resolution or mass
accuracy. It was shown before that artifactual sulfation of the hydroxyamino acids
can occur during silver staining (Gharib et al. MCP 2009, 8: 506). Here we
demonstrate that redox-active cysteines can uncontrollably be modified by nominal
80 Da, as well as 32, 48, 64, 76 and -34 Da as a result of sample preparation in
protein expression and proteomic workflows. Different reasons have been
elucidated and not all of them are fully clarified yet.
1) β-Mercaptoethanol is often used during the purification of expressed proteins
(Begg & Speicher, JBT 1999, 10: 17). It forms mixed disulfides with the protein,
which may, conclusively, be present in commercial proteins or synthetic peptides.
This is of importance to customers who rely on the activity of cysteine residues
and need them in native form and unoccupied. On the other hand, users who
study low-level protein modifications might get interferences from such
contaminants.
2) Low pKa cysteine residues are reactive and critical components in redox
signaling with sulfenic acid (Cys-SOH) being a versatile reversibly oxidized form
(Poole et al. Annu Rev Pharmacol Toxicol 2004, 44: 325). However, cysteine
oxidation may also occur during handling and proceed to irreversible modifications
such as sulfonation and the formation of dehydroalanine. This was observed at the
conditions of tryptic digestion (basic pH, 37 °C) and SDS-PAGE. Other authors
had described the phenomenon for isolated cases earlier and attributed it largely
to biological effects (Jeong et al. MCP 2011, 10(3): M110.000513; Nakanishi et al.
Biochim Biophys Acta 2004, 1698: 45). The addition of 10 mM DTT to all steps of
the work-up as well as reductive alkylation proved to be effective to prevent the
detection of the above-mentioned modifications.