A qualitative and quantitative proteomic study of human microdialysate and the cutaneous response to injury
Carolyn Gill1,2,3*, Erika Parkinson1*, Martin K Church2, Paul Skipp1, Daniel Scott4, Andrew J White4, C. David O’Connor1 and Geraldine F Clough3
Supplementary Methods
Sample preparation for GeLC-MS/MS studies
Following SDS-PAGE of dialysate proteins and staining with Colloidal Coomassie Blue, gel tracks (7 cm length) were excised, cut into 25 equal-sized pieces and subjected to in situ trypsin digestion as described (16). Briefly, gel pieces were dehydrated in acetonitrile and reduced using 10 mM DTT (final concentration) for 1 h at 56°C, The samples were subsequently alkylated in the dark using 55 mM iodoacetamide (final concentration) at room temperature. Gel pieces were washed several times by dehydrating with acetonitrile and rehydrating using 0.1 M ammonium bicarbonate. Protein digestion was accomplished by the addition of enough trypsin (1.5 ng/µl trypsin in 50 mM NH4CO3, 5 mM CaCl2)to cover the dehydrated gel pieces and incubated at 37ºC for 45 min. After incubation, 5-25 µl of the trypsin buffer without trypsin (50 mM NH4CO3, 5 mM CaCl2) was added. The samples were incubated for 16 h at 37ºC. After digestion, 15 µl of 25 mM NH4HCO3 was added to the gel pieces and incubated at 37ºC for 15 minutes with agitation. Acetonitrile was added (~ 2 times the volume of the gel pieces) and incubated for a further 15 min at 37ºC with vigorous shaking. The gel pieces were centrifuged at 9,000 x g in a bench-top microcentrifuge for 1 min and the supernatant collected. 50 µl of 5% (v/v) formic acid was added and after incubation at 37ºC for 15 min, acetonitrile added (~2 times the volume of the gel pieces) and the samples incubated for a further 15 min at 37ºC. The gel pieces were centrifuged at 9,000 x g and the supernatants pooled. Supernatants were lyophilised in vacuo and stored at +4ºC until use.
Analysis of iTRAQ data
The data from all the iTRAQ experiments were combined and subjected to further analysis. A spreadsheet was generated to show the ratio of the late to early samples (i.e. 114/116). These were log2 scaled, inversed and normalised. Outliers were removed following manual inspection and log scaling was performed to convert up- and down-regulated proteins to an equivalent scale; ratios were inverted to show the change in late sample compared to the early. Normalisation was performed using the formula “(ratio/average)/standard deviation”. This gave more consistent variance and ranges across the different iTRAQ experiments, with an average ratio of 1 and a standard deviation of 1 for the ratio in each experiment. Finally, data were averaged and the standard deviation, standard error and coefficient of variation calculated for each protein.