Data Processing, Searching, and Analysis

Quantification of protein expression was conducted using MS1 intensity based label-free quantification as described in Levin et al. (2011). Raw data were imported into Rosetta Elucidator System version 3.3 (Rosetta Biosoftware, Seattle, WA). Elucidator was used for retention time alignment and extraction of MS1 feature intensities. In parallel, database searching was performed using ProteinLynx Global Server (IdentityE) version 2.5. Database searching was carried out using the Ion Accounting algorithm described by Li et al., 2009. Trypsin was set as the protease, one missed cleavage was allowed, and fixed modification was set to carbamidomethylation of cysteines. Variable modifications included oxidation of Met.

Data were searched against a P. tricornutum protein database that was built by combining three publically available protein databases: The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/protein/?term=tricornutum), Doe Joint Genome Institute (http://genome.jgi.doe.gov/Phatr2/Phatr2.download.html), and TrEMBL (http://www.ebi.ac.uk/uniprot/database/download.html) as in Rosenwasser et al., 2014. A target-decoy strategy was performed using reversed sequences. The criteria for protein identification were set to minimum of three fragments per peptide, five fragments per protein, and minimum peptide score of 6.7, which corresponds to the false identification rate of 1%. The approach for setting the minimum identification score is based on reports by Keller et al. and termed Peptide Prophet (Keller et al., 2002; Nesvizhskii et al., 2003). Identifications were imported automatically into Elucidator for annotation of features, applying a “match between runs” approach of propagating identifications between samples. Protein quantification inference was conducted using the Hi-3 method (Silva et al., 2006). A Student’s t-test was used for statistical evaluation after logarithmic transformation of protein intensities. Fold changes were determined by dividing the arithmetic mean of the three biological replicates in each group. When a protein was detected in only one of the conditions, a fold change value of ±1,000 was assigned. All mass spectrometry data, including raw data, processed spectra, and identifications, have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner (Vizcaíno et al., 2013) with the dataset identifier PXD004694.