bace inhibitors The mass spectrometry proteomic data have

The mass spectrometry proteomic data have been deposited into the ProteomeXchange Consortium [2] via the PRIDE partner repository with the dataset identifier PXD001450.
Data set analysis of avian eggshell matrix proteins A total of 261 proteins were identified using nr NCBI database. Keratins and bovine trypsin were eliminated from the list as they appeared to be contaminants or resulting from the bace inhibitors process. Protein sequences were aligned to eliminate all redundancies. Protein groups were determined using Clustal Omega multi-alignment algorithm [7]. Sequences were blasted against nr NCBI database limited to Gallus gallus taxon using the blastp program (BLAST+suite) [8]. This was performed using R language (http://cran.r-project.org). A total of 216 non-redundant eggshell matrix protein sequences were identified. The resulting file (Supplementary Table 1) is made of EntrezGene Ids, GI numbers, protein symbols, short descriptions, information on their previous identification in the shell and in the uterine fluid, mean quantitative values and emPAI values at each time point. We compared our list with the previously published proteomes of eggshell matrix proteins [9–15]. Out of the 216 proteins, 24 proteins are novel compared to previous studies and are highlighted (Supplementary Table 1). In order to determine the potential functions of the 216 eggshell matrix proteins, 3231 GO terms were extracted from the protein sequences using GORetriever (http://agbase.msstate.edu/). CateGOrizer (http://www.animalgenome.org/tools/catego/) was then used to group the GO terms in 91 different parent term categories using a GO slim2 method. Finally, GO terms enrichments were determined using Gene set enrichment tools from Genomatix suite (www.genomatix.de). When considering GO terms associated to Molecular Function (MF) and Biological Process (BP), a total of 71 GO terms were found to be significantly enriched (p-values ranges from 9.92·10−3 to 2.96·10−7). They were grouped in 18 main categories (Table 1). Groups of importance were composed of eggshell matrix proteins involved in binding and transport activity (41 proteins) and of 27 proteins related to the biology of development. Additionally, we also reported proteins involved in response to external stimulus (13 proteins), in the regulation of biological quality (15 proteins), exhibiting enzyme regulator activity (13 proteins). Also were present proteins related to protein post-translational folding (9 proteins), homeostasis maintenance (9 proteins), multi-organism process (8 proteins), nutrition and digestion (7 proteins), the extracellular organization (4 proteins), the cell–cell adhesion (5 proteins), the oxidoreductase activity (3 proteins), the coagulation process (2 proteins), the regulation of protein phosphorylation (2 proteins), the glycerol ether metabolic process (2 proteins) and the shell calcification (2 proteins).
Quantitative dataset of avian eggshell matrix proteins In the first approach, emPAI from the proteins was calculated amongst the proteins revealed at a particular stage of shell formation at 5, 6, 7 and 16h p.o. The aim was to classify the relative abundance of the proteins within one individual stage and to determine those which were the most abundant ones. EmPAI values were reported for the 216 eggshell matrix proteins at each stage, a numerical value different from zero being introduced when the protein was present (Supplementary Table 1). EmPAI values were also used to determine the number of proteins present at a particular stage (Supplementary Table 2). The numbers of proteins showing an emPAI different from zero were 91, 132, 178 and 184 at 5, 6, 7 and 16h p.o., respectively. In the second approach, GeLC–MS/MS combined with label free quantitative analyses based on a spectral counting method were used to compare the abundances of the different proteins between the four stages of eggshell formation (Supplementary Tables 1 and 2). One way ANOVA was performed on quantitative values for protein abundance in order to reveal proteins which were at a different concentration between at least one stage relative to another one. Differences were considered to be statistically significant for p-value<0.01. Amongst the 216 proteins, 175 showed differential abundance according to the four stages of shell calcification. The mean abundance per stage of shell calcification was standardized for each protein on mean values per stage and was calculated to perform hierarchical clustering analysis. A total of ten clusters were highlighted and detailed in [1]. They were grouped in five main protein profiles associated with the different events which occur during eggshell calcification [1,3]: overabundance during the primary events of widespread deposition of amorphous calcium carbonate, totaling 27 proteins (5–6h p.o.), overabundance during transformation of ACC into crystalline calcite aggregates, totaling 13 proteins (6h p.o.), overabundance during the formation of larger calcite crystal units, totaling 47 proteins (6–7h p.o.), overabundance during rapid growth of calcite and development of a columnar structure with preferential orientation of calcite, totaling 70 proteins (16h p.o.). The last group corresponded to overabundance throughout the three late stages of calcification (6–7 to 16h p.o.), totalising 18 proteins.