Figure 1.
Two series of independent experiments were compared: (i) experiments 1 to 4 for proteins derived from HNEC cultures obtained from 4 CF NP patients vs. 4 NP patients (non CF); (ii) experiments 5 to 8 for proteins derived from HNEC cultures obtained from 4 NP patients (non CF) vs. 4 controls patients. Each couple of samples was matched randomly. For quantification of proteins, iTRAQ labeling coupled to LC-MS/MS was performed (see methods for details).
Table 1.
Description of patients.
Figure 2.
Distribution of the 186 common quantified proteins in CFNP (outer donut) and NP (inner donut) vs CTRL.
Downregulated proteins are highlighted in yellow, upregulated proteins are highlighted in red and stable proteins are highlighted in green according to the ratio thresholds (R = CF NP/CTRL or NP/CTRL). 15 proteins are upregulated in NP compared to CTRL (inner donut) and 21 proteins are upregulated in CFNP compared to CTRL (outer donut). Among them, 11 proteins are upregulated in both CFNP and NP. 20 proteins are downregulated in NP compared to CTRL (inner donut) and 48 proteins are downregulated in CFNP compared to CTRL (outer donut). Among them, 16 proteins are downregulated in both CFNP and NP. Finally, among the proteins deregulated in common between CFNP and NP, two are more regulated in CFNP and three are more downregulated in CFNP. The 42 proteins of interest for CF pathophysiology are represented in intense red and intense yellow outside the outer donut.
Figure 3.
Comparison of differentially expressed proteins in CFNP compared to NP and CTRL.
On the left, we report the ratios of 42 proteins significantly modulated in CFNP/CTRL vs NP/CTRL patients. On the right, we report proteins that are modulated in both CFNP/CTRL and NP/CTRL patients. To obtain the CFNP/CTRL ratios, relative protein expressions ratios from CF NP/NP were normalized to the average of the ratios obtained in NP vs CTRL (CF NP/CTRL = CFNP/NP×NP/CTRL).
Figure 4.
Western blot analysis of seven differentially expressed proteins.
The Western blots from the 4 NP patients and the 4 CFNP patients used for proteomic experiments are shown with their respective loading controls. A: Each protein is analyzed in three technical replicates. For normalization, the acrylamide gels were stained with Coomassie blue (total migrated protein detection) just after electro-transfer and scanned using the Odyssey LI-COR. Using the Odyssey software, protein intensity values were normalized with their corresponding loading control intensity values. B: Box plots of the intensity calculated with the Odyssey software, *p<0.01, Mann and Whitney test. (a.u.: arbitrary units). We report Maximum (max), minimum (min), first quartile (Q1), third quartile (Q3).
Figure 5.
Western blot analysis of ANXA1, STIP1, TERA, KPYM, HSPB1, EZRIN and 1433S in primary cultures of human nasal epithelial cells (HNEC) from NP (n = 4) and CFNP (n = 4).
A: Each protein was analyzed in three technical replicates. For normalization, the acrylamide gels were stained with Coomassie blue (total migrated protein detection) just after electro-transfer and scanned using the Odyssey LI-COR. Using the Odyssey software, protein intensity values were normalized with their corresponding loading control intensity values. B: Box plots of the intensity calculated with the Odyssey software, *p<0.01, Mann and Whitney test. (a.u.: arbitrary units). We report Maximum (max), minimum (min), first quartile (Q1), third quartile (Q3).
Figure 6.
Expression pattern of the 42 differential proteins of interest in CF.
The 42 differential proteins are displayed in seven different classes related to their functions (according to the AmiGO toolbox kit). Left graph: metabolism (class 1), G protein process (class 2), inflammation and oxidative stress (class 3); right graph): protein folding (class 4), proteolysis (class 5), structural protein (class 6), other (class 7). The green circle represent the absolute stable ratio value (R = 1) and the green area represent the ratio thresholds (0.73≤R≤1.37) (R = CFNP/CTRL or NP/CTRL). Scale graduations for R are 0.2 and 0.5 for left and right graph, respectively. The CFNP/CTRL ratio values of the differential proteins are represented by yellow dots (downregulated in CFNP) and red dots (upregulated in CFNP); the NP/CTRL ratio values are represented by yellow squares (downregulated in NP), red squares (upregulated in NP), and green squares (stable in NP). For the proteins in bold, the modulation was confirmed by Western blot. Protein names are from Swissprot, ratio values and p values are available in supplemental material (Table S1).
Figure 7.
Diagram of the glycolysis/pentose pathway indicating the 7 downregulated proteins in CFNP.
Figure 8.
Quantified proteins present in the CFTR physical network.
Proteomics quantitative analysis allowed us to reproducibly quantify 186 proteins. Of those, 16 could be mapped on a hand curated CFTR physical network based on the Biogrid interaction database (http://thebiogrid.org).This network is composed of 198 proteins selected according to experimental criteria for direct protein-protein interactions (surface plasmon resonance (SPR), pull-down and FRET experiments). To this bibliographic network, we have added proteins identified in our laboratory using SPR and DNA ligation assays. We built the CFTR protein interaction network by taking into account only proteins that directly interact with one another, at first and second degrees. In the graph, we represent the 16 proteins with a color code corresponding to their expression status in CF (A) and NP (B) compared to CTRL: upregulated (red), downregulated (yellow), stable (green). The network is completed by a minimal number of connecting proteins of the CFTR network (white) that could not be identified or quantified by MS/MS. Proteins that are up or downregulated in both CFNP and NP are marked by a grey star.