Mitochondrial, cytoplasmic or nuclear fractions or secretomes of uninfected control cells and influenza A virus infected cells at given timepoints were labeled with 4plex iTRAQ. The samples were first dissolved into 43 ml of iTRAQ dissolution buffer and 2 ml of each sample was run into an SDS-PAGE gel. For the intracellular fractions, equal protein amounts of each sample were taken for the iTRAQ analyses based on the silver stained gels. For secretome analyses, the whole samples were labeled. Protein alkylation, trypsin digestion and labeling of the resulting peptides were done according to manufacturer's instructions (AB Sciex). After labeling, the samples were pooled, dried and dissolved into 20 mM KH 2 PO 4 (pH 3). Labeled peptides were fractionated by strong cation exchange chromatography (SCX) on an Ettan HPLC system (Amersham Biosciences). Each SCX fraction containing labeled peptides was analysed twice with nano-LC-ESI-MS/MS using Ultimate 3000 nano-LC (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (AB Sciex) with nano-ESI ionization (approximately 22 SCX fractions for intracellular samples and 13 fractions for secretome). MS data were acquired automatically using Analyst QS 2.0 software. Information-dependent acquisition method consisted of a 0.5 s TOF-MS survey scan of m/z 400-1400. From every survey scan two most abundant ions with charge states +2 to +4 were selected for product ion scans. Once an ion was selected for MS/ MS fragmentation, it was put on an exclusion list for 60 s.