Scaffold q s v4

LC–MS of exosomal proteins was carried out using a method described previously^{60 (link),61 (link)}. Proteome Discoverer v1.4.1.114 (Thermo) was used to analyze the data collected by the mass spectrometer. The database used in Mascot v2.5.1 and SequestHT searches and the version of the Mus musculus (strain C57BL/6) proteome from Uniprot KB (Proteome ID UP000000589). In order to estimate the false discovery rate, a Target Decoy PSM Validator node was included in the Proteome Discoverer workflow. MS2 scan data from the Xcalibur RAW file were parsed for separate searches of CID and ETD MS2 scans in Mascot and Sequest, and collection of the results into a single file (.msf extension). The resulting.msf files from Proteome Discoverer were loaded into Scaffold Q + S v4.4.5 (Proteome Software, Portland, OR, USA). The scaffold was used to calculate the false discovery rate using the Peptide and Protein Prophet algorithms. Proteins were grouped to satisfy the parsimony principle.

Proteome Discoverer v2.0.0.802 (Thermo) was used to analyze the data collected by the mass spectrometer. The database used in SequestHT searches was the 7/7/2015 version of the UniprotKB Homo sapiens reference proteome canonical and isoform sequences with the nonhuman sequences from the 1/1/2012 version of thegpm.org cRAP database appended to it.
The Proteome Discover analysis and consensus workflows are included in the Supplemental Information. The analysis workflow allows for extraction of MS2 scan data from the Xcalibur RAW file and searches of CID and ETD MS2 scans in SequestHT (.msf extension). In order to estimate the false discovery rate, a Target Decoy PSM Validator node was included in the Proteome Discoverer workflow. The consensus workflow allows for collection of the data from an msf file into a result file that can be viewed in Proteome Discoverer (.pdResult extension).
The study files from Proteome Discoverer were loaded into Scaffold Q+S v4.4.5 (Proteome Software, Portland, OR, USA). The false discovery rate for peptides was calculated using the Scaffold Local FDR algorithm. Protein probabilities were calculated using the Protein Prophet algorithm. Proteins were grouped by Scaffold protein cluster analysis to satisfy the parsimony principle. The results were annotated with human gene ontology information from the Gene Ontology Annotations Database (ftp.ebi.ac.uk).