Temporal proteomic analysis of IGF-1R signalling in MCF-7 breast adenocarcinoma cells.

Dysregulation of the insulin-like growth factor 1 receptor signalling network is implicated in tumour growth and resistance to chemotherapy. We explored proteomic changes resulting from insulin-like growth factor 1 stimulation of MCF-7 adenocarcinoma cells as a function of time. Quantitative analysis using iTRAQ reagents and 2-D LC-MS/MS analysis of three biological replicates resulted in the identification of 899 proteins (p<or=0.05) with an estimated mean false-positive rate of 2.6%. Quantitative protein expression was obtained from 681 proteins. Further analysis by supervised k-means clustering identified five temporal clusters, which were submitted to the FuncAssociate server to assign overrepresented gene ontology terms. Proteins associated with vesicle transport were significantly overrepresented. We further analyzed our data set for proteins showing temporal significance using the software, extraction and analysis of differential gene expression, resulting in 20 significantly and temporally changing proteins (p<or=0.1). These significant proteins play roles in, among others, altered glucose metabolism (lactate dehydrogenase A and pyruvate kinase M1/M2) and cellular stress (nascent polypeptide-associated complex subunit alpha and heat shock (HSC70) proteins). We used multiple reaction monitoring to validate these interesting proteins and have revealed several differences in relative peptide expression corresponding to protein isoforms and variants.