Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography‐stable isotope labeling and tandem mass spectrometry

Abstract

We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.

High Precision Quantitative Proteomics Using iTRAQ on an LTQ Orbitrap: A New Mass Spectrometric Method Combining the Benefits of All

“…It should be noted that the optimal CE range for specific Q values and activation times does not seem to be a stable setting for all LTQ instruments. Different values have been published 28,32,33 and we and others 28 noticed that even on the same instrument optimal settings might fluctuate over time. However, as already mentioned, fragmentation efficiency is rather low in PQD, leading to low total ion currents in these experiments.…”

“…However, PQD suffers from very poor fragmentation efficiency and low product ion counts when compared to CID, 32,33 and is naturally less suited for precise quantitation. HCD fragmentation, on the other hand, offers high fragmentation efficiency, especially after introduction of an additional octopole dedicated for fragmentation.…”

Age-related muscle dysfunction

“…Due to the production of ROS in skeletal muscle mitochondria, which increase with age and drive these protein modifications, mitochondrial muscle proteins are particularly susceptible to carbonylation (Meany et al, 2007). The age-associated fiber-type differences are observed in the susceptibility to of proteins to carbonylation.…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…For example, one can use biotin hydrazine for the derivatisation of ketones and aldehydes, and avidin columns for specific isolation of derivatised peptides and proteins [93][94][95][96][97][98]. Interestingly, Mirzaei and Regnier [97] compared three different strategies based on biotin hydrazine tagging of carbonyls, affinity selection, proteolysis, RP-HPLC, and MS, and found that performing the affinity selection and chromatography at the protein level before proteolysis and mass spectrometric protein identification, was more informative because working with intact protein allowed the detection of crosslinked or truncated proteins.…”