Computational protocol: Quantitative Proteomic Analysis of Gingival Crevicular Fluid in Different Periodontal Conditions

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Protocol publication

[…] A statistical program (SPSS Statistics 19, IBM Brazil, São Paulo, SP, Brazil) was used for clinical analysis. Full-mouth clinical data were averaged in each patient and within groups. Clinical parameters for the 14 sampled sites were also computed for each patient and averaged within groups. Significant differences in demographic and clinical parameters among groups were determined by Kruskal-Wallis, Mann-Whitney and χ2 tests. For MS data, each survey scan (MS) was followed by automated sequential selection of seven peptides for a standard collision-induced (CID) method, with dynamic exclusion of the previously selected ions. The obtained MS/MS spectra were searched against human protein databases (Swiss Prot and TrEMBL, Swiss Institute of Bioinformatics, Geneva, Switzerland, http://ca.expasy.org/sprot/) using SEQUEST algorithm in Proteome Discoverer 1.3 software (Thermo Scientific, San Jose, CA, USA), using at least two peptides. Search results were filtered for a False Discovery rate of 1% employing a decoy search strategy utilizing a reverse database []. The proteins identified were grouped into 9 different categories based on their known biological functions.For quantitative proteome analysis, three MS raw files from each pooled clinical categories were analyzed using SIEVE software (Version 2.0 Thermo Scientific, San Jose, CA, USA). Signal processing was performed in a total of 12 MS raw files. The SIEVE experimental workflow was defined as ‘‘Control Compare Trend Analysis’’ where one class of samples are compared to one or more other classes of samples. In the present study, the HH group was compared to each of the other group (H, G and P). For the alignment step, a single MS raw file belonging to the HH group was selected as the reference file and all of the other files were adjusted to generate the best correlation to this reference file. After alignment, the feature detection and integration (or framing) process was performed using the MS level data with a feature called ‘‘Frames From MS2 Scans’’ only. When using this type of framing only MS mass-to-charge ratio (m/z) values that were associated with MS2 scan are used. Any m/z measurements that do not have MS2 were ignored. The parameters used consisted of a frame m/z width of 1500 ppm and a retention time width of 1.75 min. A total of 216,099 MS2 scans were present in all of the 12 RAW files that resulted in a total of 20,158 frames. Then peak integration was performed for each frame and these values were used for statistical analysis. Next, peptide sequences obtained from the database search using SEQUEST algorithm in Proteome Discoverer 1.3 were imported into SIEVE. A filter was applied to the peptide sequences during the import that eliminated all sequences with a Percolator q-value greater than 1% (false discovery rate). Peptides were grouped into proteins and a protein ratio and p-value were calculated, using a weighted average of the peptide intensities for the protein calculation. Only proteins observed in all four groups were quantified. HH group was used as the default group and all other three groups were compared with HH group. Relative abundance of an individual protein from HH group was considered significantly different protein level when the values observed were <0.75 for decreased abundance or >1.25 for increased abundance, and a p-value <0.05 as described previously [,].For ELISA results, mean (± standard-deviation) values were calculated for each group. Afterwards, Analysis of Variance and Student-Newman-Keuls test for pairwise comparisons was carried out to identify significant differences among groups at a 5% level. […]

Pipeline specifications

Software tools SPSS, Comet, Proteome Discoverer
Databases UniProt ExPASy
Applications Miscellaneous, MS-based untargeted proteomics
Diseases Gingivitis, Periodontal Diseases, Periodontitis, Chronic Periodontitis