Computational protocol: Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

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Protocol publication

[…] Mass spectra were analyzed by using the MaxQuant software (version 1.1.1.36). As the genomic data of sheep is incomplete, we generated a reference protein database by integrating the following databases and sequences of cow proteins and current known sheep proteins and removed duplicate proteins, including GenBank nr (20110403), Uniprot cow proteins (20110503), sheep proteins [http://www.livestockgenomics.csiro.au/sheep/] and cow proteins [http://genomes.arc.georgetown.edu/drupal/bovine/]. The MS/MS data were searched against the reference protein database using the search engine embedded in MaxQuant. Up to two missed cleavages were allowed. The first search was set to 20 ppm, and the MS/MS tolerance for CID was set to 0.5 Da. To warrant the reliability and stability of our detection platform, following criteria for inclusion/ exclusion of peptides and proteins were used, as previous studies [,]: The false discovery rate (FDR) was set to 0.01 for peptide and protein identifications. Proteins were considered identified when at least two peptides were identified and at least one of which was uniquely assignable to the corresponding sequence. Contents of the protein table were filtered to eliminate identifications from the reverse database and common contaminants. In the case of identified peptides that were all shared between two proteins, these were combined and reported as one protein group. Contents of the protein table were filtered to eliminate identifications from the reverse database and common contaminants. The minimum peptide length was set to 6 amino acids. A minimum of two peptides with one being unique was required for protein identification. To perform label-free quantification analysis, the MaxQuant software suite containing an algorithm based on the extracted ion currents (XICs) of the peptides was used. Xcalibur 2.1 (Thermo Scientific) was used as quality control program to check the quality of chromatographs. [...] In data analysis, all proteins were mapped to the Ensembl Bos taurus gene ID. The expression quantity of each protein normalized on the basis of the numbers of peptides by using MaxQuant software (version 1.1.1.36). Only peptides which is corresponding to unique proteins could use to protein quantization in the comparison between peptides and proteins. In the comparison of proteomic profiles between C and IC areas, the measured value of each biological replicate was achieved by averaging every two technical replicates of a biological replicate. Then the Student’s t-test was used to detect the significance of the differentially expressed proteins (DEPs) according to the measured value of every three biological replicates in each group, and P < 0.05 was considered significant.We used DAVID (The Database for Annotation, Visualization and Integrated Discovery) version 6.7 platform [http://david.abcc.ncifcrf.gov/] annotate biological themes for DEPs between C and IC areas. This platform often used to analyze high-throughput data [,].To assess the similarities of the different replicates, and to obtain a visual understanding of the relationship between the different areas, hierarchical clustering was carried out using CLUSTER 3.0 data analysis tool based on the clusters of protein expression profile of different technical and biological replicates. […]

Pipeline specifications

Software tools MaxQuant, DAVID
Application MS-based untargeted proteomics
Organisms Ovis aries