The Orbitrap was used to collect MS scans. All data were con verted from raw files to the. dta format using ExtractMS version 2. 0 and searched against human reference database fairly downloaded from the National Center for Biotechnology Information using the SEQUEST Sorcerer algo rithm. Searching parameters included mass tolerance of precursor ions and product ion, partial tryptic restriction, with a dynamic mass shift for oxidized Met, two maximal modification sites and a maximum of two missed cleavages. Only b and y ions were considered during the database match. To eval uate the false discovery rate, all original protein sequences were reversed to generate a decoy database that was concatenated to the original database. The FDR was estimated by the number of decoy matches and total number of assigned matches.

FDR 2 nd nt, assuming mismatches in the original database were the same as in the decoy database. To remove false positive matches, assigned peptides were grouped by a Inhibitors,Modulators,Libraries combination of trypticity and precursor ion charge state. Each group was first filtered by mass accuracy and by dynamically increasing correlation coefficient and Cn values to reduce theoretical protein FDR by the above measure to less than 1%. All MS MS spectra Inhibitors,Modulators,Libraries for proteins identified by a single peptide were manually inspected as described previously. If peptides were shared by mul tiple members of a protein family, the matched members were clustered into a single group. On the basis of the principle of parsimony, the group was represented by the protein with the greatest number of assigned peptides.

Inhibitors,Modulators,Libraries All identified Inhibitors,Modulators,Libraries proteins and ungrouped are provided respectively in Table S1 and Table S2 in Additional File 1. Quantification of peptides and proteins was based on the comparison of paired pep tides from AD and control samples. Ion current intensities for identified peptides were extracted in MS survey scans of high resolution and a ratio of the peak intensities for the peptide precursor ion was calculated using in house DQuan software as described previously. Establishing candidate biomarkers with statistical and pathway analysis Statistical analysis to evaluate the significance of the pro tein changes was performed as previously described with modifications. Relative differences in protein levels Inhibitors,Modulators,Libraries were derived from extracted ion intensities for all identi fied peptides and expressed as signal to noise ratios. A ratio of ion intensities for the peptide precursor ions from AD and control pools were calculated, log2 transformed, and averaged to obtain a protein ratio across samples. A null experiment was represented Regorafenib 755037-03-7 by a comparison of log2 transformed protein ratios for control replicates.