Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation

Abstract

Carbonylation is a non-enzymatic irreversible post-translational modification. The adduction of carbonyl groups to proteins is due to the presence of excess of ROS in cells. Carbonylation of specific amino acid side chains is one of the most abundant consequences of oxidative stress; therefore, the determination of carbonyl groups content in proteins is regarded as a reliable way to estimate the cellular damage caused by oxidative stress. This paper reports a novel RIGhT (Reporter Ion Generating Tag) (A. Amoresano, G. Monti, C. Cirulli, G. Marino. Rapid Commun. Mass Spectrom. 2006, 20, 1400) approach for selective labeling of carbonyl groups in proteins using dansylhydrazide, coupled with selective analysis by bidimensional mass spectrometry. We first applied this approach to ribonuclease A and lysozyme as model proteins. According to the so-called 'gel-free procedures', the analysis is carried out at the level of peptides following tryptic digest of the whole protein mixture. Modified RNaseA was analyzed in combined MS(2) and MS(3) scan mode, to specifically select the dansylated species taking advantage of the dansyl-specific fragmentation pathways. This combination allowed us to obtain a significant increase in signal/noise ratio and a significant increase in sensitivity of analysis, due to the reduction of duty cycle of the mass spectrometer. The unique signal obtained was correlated to peptide 1-10 of RNaseA carbonylated and labeled by dansylhydrazide. This strategy represents the first method leading to the direct identification of the carbonylation sites in proteins, thus indicating the feasibility of this strategy to investigate protein carbonylation in a proteomic approach.

Ryanodine Receptor Oxidation Causes Intracellular Calcium Leak and Muscle Weakness in Aging

“…There were no differences in the levels of nitrosylation, oxidation or PKA phosphorylation of RyR1 from muscles from mice treated with S107 vs. control. This suggests that the oxidative protein modifications are irreversible, as has been reported (Palmese et al, 2011), and protein nitrosylation may have a half-life longer than the 4 weeks of S107 treatment, which has also been reported (Hess et al, 2005). Muscles from the S107 treated mice did, however, show significantly more calstabin1 in the RyR1 complexes compared to skeletal muscle from control mice (Figures 3F and 3G).…”

“…Oxidation-dependent carbonyl modifications of proteins have been reported to be irreversible (Palmese et al, 2011). Thus, inhibiting SR Ca 2+ leak would decrease additional oxidation of the RyR1 but would not necessarily reverse the oxidation of RyR1.…”

Protein carbonylation as a major hallmark of oxidative damage: Update of analytical strategies

“…Dansyl hydrazide is an interesting tag as it generates specific reporter ions at m / z 234 and 170. The precursor ion scan mode for m / z 170 and an additional dansyl‐specific MS 3 transition ( m / z 234 → m / z 170) significantly increased the signal‐to‐noise ratio and sensitivity of the analysis (Palmese et al, ). The utility of this approach, however, for complex proteome samples has yet to be demonstrated.…”

Mass spectrometry and redox proteomics: Applications in disease

“…An example is dansylhydrazide which enhances efficiency of ionization due to its secondary nitrogens. Dansylhydrazide generates reproducible fragmentation patterns which allows MS 3 scans to be employed for localization of PCO sites in proteins (Palmese et al, ).…”