Database search
The LC-MS/MS data was searched against a junco protein database generated from a recently annotated dark-eyed junco genome. For each of the transcribed regions of the junco genome described as ”gene silhouettes,” the best scoring match among protein sequences fromGallus gallus , Taeniopygia guttata and Ficedula albicollis was identified using BLASTX program of the BLAST software version 2.9.0 allowing search against all six reading frames. Each best hit protein sequence was aligned to the corresponding gene silhouette nucleotide sequence using BLAT version 35 (Kent, 2002). The open reading frame (ORF) sequence was selected from the longest hit reported by BLAST. Proteome Discoverer version 2.1.1.21 (ThermoScientific) was used to interpret and quantify the relative amounts from the diagnostic fragment ions. The database search parameters were set as follows: two missed protease cleavage sites were allowed for trypsin digested with 5ppm precursor mass tolerance and 20 ppm for a fragment ion quantification tolerance. Trypsin was set as the protease with up to two missed cleavages allowed. Oxidation of methionine, pyroglutamine on peptide amino termini and protein N-terminal acetylation were set as variable modifications. Carbamidomethylation (C; +57Da) was set as a static modification. TMT 10plex was set as a constant modification on peptide amino termini and lysine residue side chains. Data were searched using the Sequest HT algorithm and the results were filtered via Percolator with a decoy database false discovery rate (FDR) set to < 1% as a filter for peptide identification (Spivak, Weston, Bottou, Käll, & Noble, 2009).