All “low” category proteins were discarded. The X!Tandem21 and SEQUEST22 algorithms were used for amino acid sequence ID as described.23 Quantification of proteins was carried out as described.20 Briefly, selleck products when raw files were acquired from

the LTQ mass spectrometer, all extracted ion chromatograms (XICs) were aligned by retention time. After alignment, area under the curve (AUC) for each individually aligned peak from each sample was measured, normalized, and compared for relative abundance. The current study was an exploratory “discovery” proteomics study; therefore, our study sample was not based on a formal sample size calculation. However, our sample size is generally consistent with other discovery proteomics analyses. ANOVA (analysis of variance) was used to detect significant changes in protein expression Ivacaftor manufacturer among patient groups. To eliminate technical bias, randomization of order of measurement and “quantile normalization” was used.24 Normalization was done on a log2 scale (one unit difference on this log scale is equivalent to a 2-fold change). From the ANOVA model a P-value was obtained. The P-value is an estimate of the FPR (false positive rate). The P-value was transformed to a q-value, a number that estimates the FDR (false discovery rate). The P-value threshold was fixed to control the FDR at 5% (<0.05).

A protein with a “significant change” or “differential expression” was defined as a difference in protein expression between any two patient groups with a q-value < 0.05. For each protein a separate ANOVA model was fit using PROC MIXED in SAS software (SAS Institute, Cary, NC): Positive fold changes (FC), when mean treated group ≥ mean control group, were computed from the means on the AUC scale (antilog): FC = mean treated group/mean control group. Negative FCs, when mean control group > mean treated group, were computed from the means on the AUC scale (antilog): FC = −mean treated group/mean control group. Absolute Interleukin-3 receptor (positive)

values of the FCs were computed. The median percent coefficient of variation (%CV) for each priority level was determined by dividing the standard deviation (SD) by the mean on the AUC scale and is given on a percent scale. Only priority 1 proteins with a significant change (q < 0.05) between any two patient groups were considered for further analyses (72 proteins). However, the maximum observed change in the mean log2 intensity for the internal standard (chicken lysozyme) between groups was 14% (1.14-fold change); therefore, only priority 1 proteins with a significant change >14% (q < 0.05) were considered for characterization of biological function (56 proteins). In order to further evaluate priority 1 proteins as biomarker candidates, more stringent criteria were applied to discriminate between groups. For these analyses, only priority 1 proteins with a significant change >30% between any two groups (q < 0.05) were considered (27 proteins).