Introduction Aberrant expression of oncogenic signalling proteins and their activation by phosphorylation is a key feature of malignancy. Current methodologies do not allow detailed analysis of the phosphorylation patterns of oncoproteins in small clinical samples. Therefore we developed an exquisitely sensitive capillary is oelectric focusing immunoassay (cIEF) method for discriminating phosphorylated isoforms of ERK and AKT in lung tissue.

Methods Participants undergoing curative resection for early stage NSCLC were recruited. Samples from normal lung and tumour tissue were taken shortly after resection and analysed using cIEF.

Results Twenty patients were recruited (12 male, 8 female), mean age 67.3 years (SD 7.5), 10 current and 10 former smokers with a mean smoking exposure of 48.4 pack years (SD 24.4). Tumour tissue (9 squamous and 11 adenocarcinoma) was collected and paired macroscopically normal lung that was histologically normal (n=7) or showed emphysema (n=13), graded pathologically as mild (n=7), moderate (n=3) or severe (n=3). The coefficient of variance was 7.6% for ERK and 4.2% for the AKT assay. The limits of detection and quantification were 8ng and 16ng of total protein respectively.

Diphosphorylated ERK (active form) was nearly three-fold higher in emphysematous lung tissue than normal lung tissue (p=0.002) and was associated with pathological severity of emphysema. In contrast, diphosphorylated ERK did not differ between paired normal lung and tumour (p=0.45) and there was no correlation of ERK status with age, gender, smoking status, tumour site, histology or stage. AKT was significantly more phosphorylated in tumour than matched normal lung. There was a trend for more phosphorylated AKT with poor differentiation but no correlation with tumour histology, size, site or stage of disease. Current smokers had more phosphorylated AKT than ex-smokers. There was no difference in the phosphorylation patterns of AKT according to age, gender or emphysema status.

Conclusions We found a strong relationship between diphosphorylated ERK and emphysema. By contrast, AKT phosphostatus was associated with lung cancer but not with emphysema. The requirement for just nanograms of protein defines the potential of this technique for determining the phosphostatus of oncogenic signalling proteins in tiny clinical samples.