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).