ICAT-MS-MS time course analysis of atrophying mouse skeletal muscle cytosolic subproteome.

Skeletal muscle atrophy is a process in which protein degradation exceeds protein synthesis, resulting in a decrease of the muscle's physiological cross-sectional area and mass, and is often a serious consequence of numerous health problems. We used the isotope-coded affinity tag (ICAT) labelling approach and MS-MS to protein profile cytosolic subcellular fractions from mouse tibialis anterior skeletal muscle undergoing 0, 4, 8, or 16 days of immobilisation-induced atrophy. For the validation of peptide and protein identifications statistical algorithms were applied to the sequence database search results in order to obtain consistent sensitivity/error rates for protein and peptide identifications at each immobilisation time point. In this study, we identified and quantified a large number of mouse skeletal muscle proteins. At a protein probability (P) of P> or = 0.9 (corresponding to a false positive error rate of less than 1%) 807 proteins were identified (231, 226, 217 for 4, 8, 16 days of immobilisation and 133 for the control sample, respectively), from which 51 displayed altered protein abundance with atrophy. Due to randomness of data acquisition, a full time course could be generated only for 62 proteins, most of which displayed unchanged protein abundance. In spite of this, useful information about dataset characteristics and underlying biological processes could be obtained through gene over-representation analysis. 20 gene categories-mainly but not exclusively encoded by the subset of overlapping proteins--were consistently found to be significantly (p < 0.05) over-represented in all 4 sub-datasets.

Quantitative proteomics identifies oxidant-induced, AtMPK6-dependent changes in Arabidopsis thaliana protein profiles.

In Arabidopsis thaliana, oxidant-induced signalling has been shown to utilize the mitogen-activated protein kinase (MAPK), AtMPK6. To identify proteins whose accumulation is altered by ozone in an AtMPK6-dependent manner we employed isotope-coded affinity tagging (ICAT) technology to investigate the impact of AtMPK6-suppression on the protein profiles in Arabidopsis both before (air control) and during continuous ozone (O(3)) fumigation (500 nL L(-1) for 8 h). Among the 150 proteins positively identified and quantified in the O(3)-treated plants, we identified thirteen proteins whose abundance was greater in the AtMPK6-suppressed genotype than in wild-type (WT). These include the antioxidant proteins, monodehydroascorbate reductase, peroxiredoxin Q, and glutathione reductase. A further eighteen proteins were identified whose abundance was lower in the ozone-treated AtMPK6-suppressed line relative to ozone-exposed WT plants. These predominantly comprised proteins involved in carbohydrate-, energy-, and amino acid metabolism, and tetrapyrrole biosynthesis. In control plants, five proteins increased, and nine proteins decreased in abundance in the AtMPK6-suppressed genotype compared to that of the WT, reflecting changes in the protein composition of plants that have AtMPK6 constitutively suppressed. Since a number of these proteins are part of the redox response pathway, and loss of AtMPK6 renders Arabidopsis more susceptible to oxidative stress, we propose that AtMPK6 plays a key role in the plant's overall ability to manage oxidative stress.