TY - JOUR
T1 - Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography-stable isotope labeling and tandem mass spectrometry
AU - Meany, Danni L.
AU - Xie, Hongwei
AU - Thompson, La Dora V.
AU - Arriaga, Edgar A.
AU - Griffin, Timothy J.
PY - 2007/4
Y1 - 2007/4
N2 - 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.
AB - 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.
KW - Mitochondria
KW - Protein carbonylation
KW - Pulsed Q dissociation
KW - Stable isotope labeling
KW - iTRAQ
UR - http://www.scopus.com/inward/record.url?scp=34247464726&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34247464726&partnerID=8YFLogxK
U2 - 10.1002/pmic.200600450
DO - 10.1002/pmic.200600450
M3 - Article
C2 - 17390297
AN - SCOPUS:34247464726
SN - 1615-9853
VL - 7
SP - 1150
EP - 1163
JO - Proteomics
JF - Proteomics
IS - 7
ER -